NMDAR antagonist for the treatment of pervasive development disorders

ABSTRACT

A method of treating a pervasive development disorder in a subject includes administering to the subject an amount of an NMDAR antagonist effective to ameliorate biochemical and functional abnormalities in the subject associated with loss-of-function mutations of the gene encoding methyl-CpG binding protein 2 (MeCP2).

RELATED APPLICATION

This application claims priority from U.S. Provisional Application Nos.61/756,739, filed Jan. 25, 2013 and 61/810,974 filed Apr. 11, 2013, thesubject matter of which are incorporated herein by reference in theirentirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. NS057398awarded by The National Institutes of Health/NINDS, Autism SpeaksFoundation. The United States government has certain rights to theinvention.

FIELD OF THE INVENTION

The present application relates to compositions and methods used fortreating pervasive development disorders using NMDA receptorantagonists.

BACKGROUND

Pervasive development disorders refer to a group of disorderscharacterized by delays in the development of multiple basic functionsincluding socialization and communication. Pervasive developmentaldisorders include autism, Asperger syndrome, pervasive developmentaldisorder not otherwise specified (PDD-NOS), childhood disintegrativedisorder, and Rett syndrome.

Rett Syndrome (RTT) is a pervasive developmental disorder that sharesseveral clinical signs with autism but has an unrelated cause. RTT iscaused by loss-of-function mutations in the gene encoding the methyl-CpGbinding protein 2 (MeCP2), a transcriptional regulatory protein. After aperiod of apparently normal early postnatal development, RTT patientsdevelop a spectrum of symptoms that generally includes loss of acquiredspeech, head growth deceleration, autistic behaviors, motor, respiratoryand autonomic dysfunction and increased risk of seizures. Inactivationof Mepc2 at any age leads to RTT-like symptoms indicating that MeCP2protein is required across the lifespan for normal brain function.

However, loss of MeCP2 in RTT patients and mouse models is notassociated with neuronal cell death or axonal degeneration, althoughneurons are smaller, more densely packed than normal and exhibit reduceddendritic arborizations, spine density and synapse number. In addition,Mecp2 mutant mice exhibit defects in neuronal and synaptic function,including alterations in excitatory/inhibitory (E/I) balance. Thesemicrocircuit abnormalities are accompanied by altered expression ofneurotransmitters, neurotransmitter synthesizing enzymes, receptors andtransporters, as well as molecules required for synapse development.

SUMMARY

Embodiments described herein relate to compositions and methods oftreating pervasive development disorders, such as Rett Syndrome, in asubject. The method includes administering to the subject atherapeutically effective amount of an NMDAR antagonist. Thetherapeutically effective amount is an amount effective to amelioratebiochemical and functional abnormalities associated withloss-of-function mutations of the gene encoding methyl-CpG bindingprotein 2 (MeCP2) in the subject. In some embodiments, thetherapeutically effective amount of an NMDAR antagonist can besub-anesthetic.

In an aspect of the invention the NMDAR antagonist is selected from thegroup consisting of Amantadine, AZD6765, Dextrallorphan,Dextromethorphan, Dextrorphan, Diphenidine, Dizocilpine (MK-801),Ethanol, Eticyclidine, Gacyclidine, Ibogaine, Memantine, Methoxetamine,Nitrous oxide, Phencyclidine, Rolicyclidine, Tenocyclidine, Methoxydine,Tiletamine, Xenon, Neramexane, Eliprodil, Etoxadrol, Dexoxadrol,WMS-2539, NEFA, Delucemine, 8A-PDHQ, Aptiganel, HU-211, Remacemide,Rhynchophylline, Ketamine, 1-Aminocyclopropanecarboxylic acid (ACPC),7-Chlorokynurenate′ DCKA (5,7-dichlorokynurenic acid), Kynurenic acid,Lacosamide, L-phenylalanine, Neurotransmitters, Psychedelics, Long-termpotentiation, and NMDA. In some embodiments, the NMDAR antagonist isRemacemide.

In some embodiments, the method further includes administering to thesubject a therapeutically effective amount of a TrkB agonist. The TrkBagonist can be selected from the group consisting of a small molecule,protein and an antibody. In some embodiments the TrkB agonist is anampakine. The ampakine can include an allosteric modulator of theAMPA-receptor.

In a particular embodiment, the NMDAR antagonist can include Remacemideand the TrkB agonist can includeN,N′,N″Tris(2-hydroxyethyl)-1,3,5-benzenetricarboxamide (LM22A-4).

Other embodiments described herein relate to a pharmaceuticalcomposition for the treatment of Rett syndrome. The composition includesa therapeutically effective amount of an NMDAR antagonist, atherapeutically effective amount of a TrkB agonist, and apharmaceutically acceptable diluent or carrier. The therapeuticallyeffective amount of an NMDAR antagonist and a TrkB agonist can be theamount effective to ameliorate biochemical and functional abnormalitiesassociated with loss-of-function mutations of the gene encodingmethyl-CpG binding protein 2 (MeCP2) in a subject having Rett syndrome.In some embodiments, the therapeutically effective amount of an NMDARantagonist can be sub-anesthetic.

In some embodiments, the NMDAR antagonist can be selected from the groupconsisting of Amantadine, AZD6765, Dextrallorphan, Dextromethorphan,Dextrorphan, Diphenidine, Dizocilpine (MK-801), Ethanol, Eticyclidine,Gacyclidine, Ibogaine, Memantine, Methoxetamine, Nitrous oxide,Phencyclidine, Rolicyclidine, Tenocyclidine, Methoxydine, Tiletamine,Xenon, Neramexane, Eliprodil, Etoxadrol, Dexoxadrol, WMS-2539, NEFA,Delucemine, 8A-PDHQ, Aptiganel, HU-211, Remacemide, Rhynchophylline,Ketamine, 1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate′DCKA (5,7-dichlorokynurenic acid), Kynurenic acid, Lacosamide,L-phenylalanine, Neurotransmitters, Psychedelics, Long-termpotentiation, and NMDA. In other embodiments, the NMDAR antagonist isRemacemide.

In a further aspect, the TrkB agonist can be selected from the groupconsisting of a small molecule, protein and an antibody. In someembodiments the TrkB agonist is an ampakine. The ampakine can include anallosteric modulator of the AMPA-receptor.

In some aspects, the pharmaceutical composition further includes atherapeutically effective amount of a GABAR agonist.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates photographs showing immunostains of reduced Fosexpression in forebrain and midbrain structures in symptomatic Mecp2Null mice. At 6 weeks of age, Null mice (A3-F4) exhibit markedly reducedlevels of Fos expression in a discrete subset of cortical andsubcortical structures, including the prelimbic and infralimbic cortices(A), cingulated cortex (B), retrosplenial cortex (C), piriform cortex(D), the nucleus accumbens (E), and periaqueductal gray (F) comparedwith Wt (A1-F2). A2-F2 and A4-F4 show higher-magnification views of thesections shown in A1-F1 and A3-F3, respectively. ac, Anteriorcommissure; cc, corpus callosum.

FIG. 2 illustrates photomicrographs and graphical illustrations showingincreased Fos expression in the hindbrain nTS in symptomatic Mecp2 Nullmice. At 6 weeks of age, Null mice exhibit markedly increased levels ofFos expression in subnuclei within the nucleus tractus solitarius (nTS)of the medulla. A, Photomicrographs of representative coronal sectionsthrough nTS at 2 different rostrocaudal levels, with a schematic overlayillustrating nTS subnuclei and landmarks. B, Quantitative analysis ofgenotype-dependent differences in Fos expression across the entirerostrocaudal extent of the lnTS, mnTS, and nComm subnuclei of nTS inincrements of 80 μm. Note that except for the most anterior andposterior sections through nComm, Fos expression is significantlyelevated throughout the rostrocaudal extent of the Null nTS comparedwith Wt (p<0.05). Dotted lines mark the obex. C, Average counts ofFos-positive cells at each level were combined to provide an estimate ofthe absolute total number of Fos-positive cells throughout therostrocaudal extent of nTS in Null vs. Wt. 10N, Dorsal motor nucleus ofvagus; 12N, hypoglossal nucleus; AP, area postrema; lnTS, lateral nTS;mnTS, medial nTS; nComm, commissural nTS; TS, tractus solitarius.*p<0.05; ***p<0.001.

FIG. 3 illustrates graphical representations of exaggerated evokedsynaptic transmission in the adult Null lnTS and nComm A, EPSCs evokedin the lnTS by low-frequency TS stimulation have significantly largeramplitudes in the Nulls compared with Wt. B, Statistical summary ofgenotype-dependent differences in eEPSC amplitudes in lnTS and nComm C,Synaptic depression evoked by 20 Hz stimulus trains is unaffected byMecp2 genotype. D, eEPSC amplitudes averaged across the entire 20 Hzstimulus train are significantly larger in the Null lnTS compared withWt, with a similar trend in nComm E, Input-output curve demonstratingthat regardless of stimulus intensity, based on the individual neurons'stimulation response threshold, eEPSC amplitudes are larger in Nullscompared with Wt. *p<0.05; **p<0.01; ***p<0.001.

FIG. 4 illustrates graphical representations of enhanced spontaneousexcitatory currents at primary afferent synapses in the Null nTScompared with Wt. A, Representative recordings from a Wt and a Null lnTSsecond-order neuron illustrating higher sPSC frequency in the Nulls. B,Group data reveal a significantly higher sPSC frequency in both NulllnTS and nComm compared with Wt. C, Representative recordings from a Wtand a Null lnTS second order neuron at low (top traces) and high (bottomtraces) resolution illustrating higher mEPSC frequency and amplitudes inthe Nulls. D, E, Cumulative mEPSC frequency (D) and amplitude (E)distribution curves from recordings of lnTS and nComm neurons showright-shifts in the Nulls, indicating higher mEPSC frequencies andamplitudes, respectively, compared with Wt. *p<0.05; **p<0.01;***p<0.001.

FIG. 5 illustrates graphical representations of evoked synaptictransmission is normal in the Null nTS at 3 weeks of age despite reducedspontaneous excitatory currents. A, B, 0.5 and 20 Hz stimulation bothyield similar EPSC amplitudes in juvenile Wt and Null lnTS second-orderneurons. C, D, Spontaneous PSC frequency is lower in juvenile Null lnTSneurons compared with Wt. E, Raw traces illustrating reduced mEPSCfrequencyandamplitudesintheNullnTSat3 weeks. F, G, Cumulative mEPSCfrequency (F) and amplitude (G) distribution curves demonstrate leftshifts in the Nulls at 3 weeks, indicating lower mEPSC frequencies andlower amplitudes, respectively, compared with Wt. *p<0.05; **p<0.01;***p<0.001.

FIG. 6 illustrates photomicrographs and graphical illustrationsillustrating systemic treatment with ketamine acutely increases Fosexpression in the forebrain of Wt and Null mice and reverses theFos-deficient phenotype in Nulls. A, B, Injection of ketamine (100mg/kg, i.p.) increases Fos expression throughout the forebrain in Nullsand Wt; representative sections from the prelimbic and infralimbiccortices (A) and cingulate cortex (B) are shown. C, Ketamine causes adose-dependent increase in Fos expression levels in Null mice, restoringFos expression to naive Wt levels as shown here in the piriform cortex.In Wt, 100 mg/kg ketamine increased the number of Fos-positive cells by20%. *p<0.05; **p<0.01; ***p<0.001.

FIG. 7 illustrates a graphical illustration of Het mice exhibit abnormalprepulse inhibition of acoustic startle which is restored to Wt levelsby acute treatment with a sub-psychotomimetic dose of ketamine. Het miceexhibit a significant increase in PPI amplitude at 11 weeks of age thatis restored to Wt levels by acute treatment with ketamine at 8 mg/kg.*p<0.05; ***p<0.001.

FIG. 8 is graphical illustrations of the development of respiratorydysfunction in 8- to 12-week-old Mecp2^(−/+) (Het) mice. A1-A4,Breathing frequency (A1), T_(tot) (A2), T_(i) (A3), and T_(e) (A4) arenot significantly different between Wt(open bars) and Het (gray bars)mice at 8 weeks of age (Wt, n=7; Het, n=5). Significant increases infrequency, associated with decreased T_(i), T_(e), and T_(tot), areobserved in 10-week-old Hets compared with Wt controls (Wt, n=22; Het,n=14); these differences persist at 12 weeks (Wt, n=16; Het, n=14) withthe exception of T_(i). Results are expressed as the mean in percentageWt±SEM. *p<0.05, **p<0.01, ***p<0.001, unpaired t test. B1,Box-and-whisker plots showing the number of apneas in Wt (open bars) andHet (gray bars) mice at 8, 10, and 12 weeks of age. B2, The proportionof Hets exhibiting significantly more apneas than Wt increased from 20%at 8 weeks to 50% at 12 weeks of age.

FIG. 9 illustrates BDNF protein levels in 8-, 10-, and 12-week-old Mecp2Het mice. A-C, BDNF levels, measured by ELISA, in the NG (A), medulla(B), and pons (C) of Wt (open bars) and Het mice (gray bars). Resultsare expressed as the mean in percentage Wt±SEM (n=5-19 NG per group).*p<0.05, **p<0.01, ***p<0.001, unpaired t test. D, BDNF immunostaining,representative of 3 Wt and 3 Het littermate pairs, demonstrates reducedBDNF levels in the nTS subregion of the dorsomedial medulla in12-week-old Het animals compared with Wt. AP, area postrema; tS, tractussolitarius.

FIG. 10 is a graphical illustration showing treatment of Mecp2 Het micewith LM22A-4 restores normal respiratory frequency by increasing T_(e)and T_(tot) Animals were treated from 8 to 12 weeks of age as describedin Materials and Methods. A, Representative plethysmographic tracesshowing the breathing pattern of Wt vehicle-treated, Wt LM22A-4-treated,Het vehicle-treated, and Het LM22A-4-treated mice. B, Comparison of thebreathing frequency, T_(tot), T_(i), and T_(e) among all four treatmentgroups. Results are expressed as the mean in percentage Wt±SEM (Wtvehicle-treated, n=29, open bars; Wt LM22A-4 treated, n=13, light graybars; Het vehicle-treated, n=22, dark gray bars; Het LM22A-4-treated,n_22, black bars). *p<0.05, **p<0.01, ***p<0.001, ANOVA I with post hocLSD test. C, Body weight is unaffected by 5 weeks of treatment withLM22A-4 (50 mg/kg, i.p., b.i.d.) Results are shown as mean±SEM (Wtvehicle-treated, n=26, black cross marker; Wt LM22A-4 treated, n=11,dark circle markers; Het vehicle-treated, n=24, open square markers; HetLM22A-4 treated, n_29, dark triangle markers).

FIG. 11 illustrates TrkB phosphorylation deficits in Mecp2 Het mice arereversed by chronic treatment with LM22A-4. Top, Representative Westernblots showing phosphorylated TrkB Y817 (p TrkB), full-length TrkB(TrkB-F), truncated TrkB (TrkB-T), and actin in medulla and pons samplesfrom (left to right) Wt vehicle-treated, Wt drug-treated, Hetvehicle-treated, and Het drug-treated mice. Graphs, Summary resultsshowing the ratios of p-TrkB/TrkB-F, p-TrkB F/actin, TrkB-F/actin andTrkB-T/actin, respectively, in medulla and pons samples from all fourtreatment groups [open bars, Wt vehicle-treated (Wt+V); light gray bars,Wt drug-treated (Wt+D); dark gray bars, Het vehicle-treated (Het+V);black bars, Het drug-treated (Het+D)]. Results are expressed as themean±SEM (medulla; Wt+V, n=9; Wt+D, n=3; Het+V, n=8; Het+D, n=13; pons;Wt+V, n=13; Wt+D, n=7; Het+V, n=11; Het+D, n=15). *p<0.05, **p<0.01,***p<0.001, ANOVA I with post hoc LSD test.

FIG. 12 illustrates AKT phosphorylation deficits in the pons of Mecp2Het mice are reversed by chronic treatment with LM22A-4. Top,Representative Western blots showing p-AKT, total AKT (AKT), p-ERK,total ERK (ERK), and actin in pons samples from (left to right in eachblot) Wt+V, Wt+D, Het+V, and Het+D mice. Graphs, Summary results showingthe ratios of p-AKT/AKT, p-ERK/ERK, p-AKT/actin, p-ERK/actin, AKT/actinand ERK/actin in pons samples from all four treatment groups (open bars,Wt+V; light gray bars, Wt_D; dark gray bars, Het+V; black bars, Het+D).Results are expressed as the mean±SEM (Wt+V, n=13; Wt+D, n=7; Het+V,n=11; Het+D, n=14). *p<0.05, **p<0.01, ***p<0.001, ANOVA I with post hocLSD test.

FIG. 13 illustrates AKT and ERK phosphorylation in the medulla. Top,Representative Western blots showing p-AKT, total AKT (AKT),p-ERK, totalERK (ERK) and actin in medulla samples from (left to right in each blot)Wt+V, Wt+D, Het+V, and Het+D mice. Graphs, Summary results showing theratios of p-AKT/AKT, p-ERK/ERK, p-AKT/actin, p-ERK/actin, AKT/actin andERK/actin in medulla samples from all four treatment groups (open bars,Wt+V; light gray bars, Wt+D; dark gray bars, Het+V; black bars, Het+D).Results are expressed as the mean_SEM (Wt+V, n=9; Wt+D, n=3; Het+V, n=8;Het+D, n=13). *p<0.05, **p<0.01, ***p<0.001, ANOVA I with post hoc LSDtest.

DETAILED DESCRIPTION

The present invention is not limited to the particular methodology,protocols, and reagents, etc., described herein and as such may vary.The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which is defined solely by the claims. Other than in theoperating examples, or where otherwise indicated, all numbers expressingquantities of ingredients or reaction conditions used herein should beunderstood as modified in all instances by the term “about.”

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

Unless otherwise defined, scientific and technical terms used hereinshall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures utilized in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridizationdescribed herein are those well known and commonly used in the art.

As used herein, the term “administering” to a patient includesdispensing, delivering or applying an active compound in apharmaceutical formulation to a subject by any suitable route fordelivery of the active compound to the desired location in the subject(e.g., to thereby contact a brain stem and forebrain neurons), includingadministration into the cerebrospinal fluid or across the blood-brainbarrier, delivery by either the parenteral or oral route, intramuscularinjection, subcutaneous or intradermal injection, intravenous injection,buccal administration, transdermal delivery and administration by therectal, colonic, vaginal, intranasal or respiratory tract route.Specific routes of administration may include intraperitoneal (i.p.)injection and/or via oral routes.

As used herein an “effective amount” of an agent or combination ofagents is an amount sufficient to achieve a desired therapeutic orpharmacological effect, such as an amount that is capable ofameliorating biochemical and functional abnormalities associated withloss-of-function mutations of the gene encoding methyl-CpG bindingprotein 2 (MeCP2). An effective amount of an agent or combinatorytherapy as defined herein may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of theagent to elicit a desired response in the subject. Dosage regimens maybe adjusted to provide the optimum therapeutic response. An effectiveamount is also one in which any toxic or detrimental effects of theactive compound are outweighed by the therapeutically beneficialeffects.

As used herein, the term “therapeutically effective amount” refers tothat amount of a composition that results in amelioration of symptoms ora prolongation of survival in a patient. A therapeutically relevanteffect relieves to some extent one or more symptoms of a disease orcondition or returns to normal either partially or completely one ormore physiological or biochemical parameters associated with orcausative of the disease or condition, e.g., Rett syndrome.

As used herein, the terms “patient” and “subject” refer to any animal,including, but not limited to, humans and non-human animals (e.g.,rodents, arthropods, insects, fish (e.g., zebrafish), non-humanprimates, ovines, bovines, ruminants, lagomorphs, porcines, caprines,equines, canines, felines, ayes, etc.), which is to be the recipient ofa particular treatment. Typically, the terms “host,” “patient,” and“subject” are used interchangeably herein in reference to a humansubject.

As used herein, the terms “subject suffering from Rett syndrome”,“subject having Rett syndrome” or “subjects identified with Rettsyndrome” refers to subjects that are identified or diagnosed as havingor likely having a loss-of-function mutation in the gene encoding themethyl-CpG binding protein MeCP2 gene, which causes Rett syndrome.

The term “modulate,” as used herein, refers to a change in thebiological activity of a biologically active molecule. Modulation can bean increase or a decrease in activity, a change in bindingcharacteristics, or any other change in the; biological, functional, orimmunological properties of biologically active molecules.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments consist of, but are not limited to,test tubes and cell culture. The term “in vivo” refers to the naturalenvironment (e.g., an animal or a cell) and to processes or reactionthat occur within a natural environment.

A “known therapeutic compound” refers to a therapeutic compound that hasbeen shown (e.g., through animal trials or prior experience withadministration to humans) to be effective in such treatment orprevention.

“Treating” or “treatment” of a condition or disease includes: (1)preventing at least one symptom of the conditions, i.e., causing aclinical symptom to not significantly develop in a mammal that may bepredisposed to the disease but does not yet experience or displaysymptoms of the disease, (2) inhibiting the disease, i.e., arresting orreducing the development of the disease or its symptoms, or (3)relieving the disease, i.e., causing regression of the disease or itsclinical symptoms. Treatment, prevention and ameliorating a condition,as used herein, can include, for example decreasing or eradicating adeleterious or harmful condition associated with Rett syndrome. Examplesof such treatment include: decreasing breathing abnormalities,decreasing motor dysfunction, and improving respiratory and neurologicalfunction.

“Cyano” refers to the group —CN.

“Halogen” or “halo” refers to fluorine, bromine, chlorine, and iodineatoms.

“Hydroxy” refers to the group —OH.

“Thiol” or “mercapto” refers to the group —SH.

“Sulfamoyl” refers to the —SO₂NH₂.

“Alkyl” refers to a cyclic, branched or straight chain, alkyl group ofone to eight carbon atoms. The term “alkyl” includes reference to bothsubstituted and unsubstituted alkyl groups. This term is furtherexemplified by such groups as methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl,cyclohexyl, i-amyl, n-amyl, and hexyl. Substituted alkyl refers to alkylas just described including one or more functional groups such as aryl,acyl, halogen, hydroxyl, amido, amino, acylamino, acyloxy, alkoxy,cyano, nitro, thioalkyl, mercapto and the like. These groups may beattached to any carbon atom of the lower alkyl moiety. “Lower alkyl”refers to C₁-C₆ alkyl, with C₁-C₄ alkyl more preferred. “Cyclic alkyl”includes both mono-cyclic alkyls, such as cyclohexyl, and bi-cyclicalkyls, such as bicyclooctane and bicycloheptane. “Fluoroalkyl” refersto alkyl as just described, wherein some or all of the hydrogens havebeen replaced with fluorine (e.g., —CF₃ or —CF₂CF₃).

“Aryl” or “Ar” refers to an aromatic substituent which may be a singlering or multiple rings which are fused together, linked covalently, orlinked to a common group such as an ethylene or methylene moiety. Thearomatic ring(s) may contain a heteroatom, such as phenyl, naphthyl,biphenyl, diphenylmethyl, 2,2-diphenyl-1-ethyl, thienyl, pyridyl andquinoxalyl. The term “aryl” or “Ar” includes reference to bothsubstituted and unsubstituted aryl groups. If substituted, the arylgroup may be substituted with halogen atoms, or other groups such ashydroxy, cyano, nitro, carboxyl, alkoxy, phenoxy, fluoroalkyl and thelike. Additionally, the aryl group may be attached to other moieties atany position on the aryl radical which would otherwise be occupied by ahydrogen atom (such as 2-pyridyl, 3-pyridyl and 4-pyridyl).

The term “alkoxy” denotes the group —OR, where R is lower alkyl,substituted lower alkyl, aryl, substituted aryl, aralkyl or substitutedaralkyl as defined below.

The term “acyl” denotes groups —C(O)R, where R is alkyl, substitutedalkyl, alkoxy, aryl, substituted aryl, amino and alkylthiol.

“Carbocyclic moiety” denotes a ring structure in which all ring verticesare carbon atoms. The term encompasses both single ring structures andfused ring structures. Examples of aromatic carbocyclic moieties arephenyl and naphthyl.

“Heterocyclic moiety” denotes a ring structure in which one or more ringvertices are atoms other than carbon atoms, the remainder being carbonatoms. Examples of non-carbon atoms are N, O, and S. The termencompasses both single ring structures and fused ring structures.Examples of aromatic heterocyclic moieties are pyridyl, pyrazinyl,pyrimidinyl, quinazolyl, isoquinazolyl, benzofuryl, isobenzofuryl,benzothiofuryl, indolyl, and indolizinyl.

The term “amino” denotes the group NRR′, where R and R′ mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl as defined below or acyl.

The term “amido” denotes the group —C(O)NRR′, where R and R′ mayindependently be hydrogen, lower alkyl, substituted lower alkyl, aryl,substituted aryl as defined below or acyl.

The term “independently selected” is used herein to indicate that thetwo R groups, R¹ and R², may be identical or different (e.g., both R¹and R² may be halogen or, R¹ may be halogen and R² may be hydrogen,etc.).

Embodiments described herein relate to compositions and methods oftreating pervasive development disorders, such as Rett Syndrome orautism spectrum disorder, in a subject. It was found that loss offunction mutations in Mecp2, the gene mutated in Rett syndrome (RTT),disrupts the balance of excitation and inhibition between the forebrainand brainstem in mouse models of the disease. Without being bound bytheory, it is believed that perturbations of circuit function within andbetween the forebrain and brainstem are at least partially responsiblefor the pathophysiology of pervasive development disorders, such as RTT,i.e., the combination of deficits (hypoactivity) in forebrain-mediatedfunctions, such as cognition, social communication and motor control onthe one hand, and dysregulation (hyperexcitability) ofbrainstem-mediated functions, such as breathing, on the other.

Using Fos protein as a surrogate marker of neuronal activity, it wasfound that RTT mice exhibit reduced activity throughout the forebrain,including reduced activity in structures critical for sensorimotorprocessing, cognition, motor control and social communication. It wasalso found that treatment of RTT mice with a low, sub-anesthetic dose ofan N-methyl-D-aspartate receptor (NMDAR) antagonist acutely reverseshypoactivity in forebrain circuits and significantly improves at leastone measure of forebrain function, i.e., prepulse inhibition of acousticstartle without altering brainstem hyperactivity.

In some embodiments, the NMDAR antagonist can be administered to asubject having or suspected of having a pervasive development disorder,such as RTT or autism spectrum disorder, to prevent, ameliorate orreverse pathologies associated with MeCP2 loss by reversing forebrainhypoactivity. Accordingly, a method for treating a pervasive developmentdisorder, such as RTT, can include administering an NMDAR antagonist toa subject such that when administered ameliorates the core neurologicalsymptoms of a pervasive development disorder, such as RTT, byre-establishing the normal balance between excitation and inhibitionwithin and between the forebrain and brainstem, respectively.

In some embodiments, the NMDAR antagonist can include an agent capableof antagonizing, or inhibiting the action of, the N-Methyl-D-aspartatereceptor. Examples of NMDAR antagonists include, but are not limited to:NMDA site antagonists, such as DL-AP7 (DL-2-Amino-7-phosphonoheptanoicacid), DL-AP5 (DL-2-Amino-5-phosphonopentanoic acid), D-AP5(D-(−)-2-Amino-5-phosphonopentanoic acid), L-AP5(L-(+)-2-Amino-5-phosphonopentanoic acid), D-AP7(D-(−)-2-Amino-7-phosphonoheptanoic acid), (RS)-CPP((RS)-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid), (R)-CPP(3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid), (R)-4CPG((R)-4-Carboxyphenylglycine), LY 235959([3S-3a,4aa,6b,8aa)]-Decahydro-6-(phosphonomethyl)3isoquinolinecarboxylicacid)), ((CGS-19755); Competitive NMDA antagonists such as: selfotel CGS19755 (cis-4-[Phoshomethyl]-piperidine-2-carboxylic acid), SDZ 220-581((S)-a-Amino-2′-chloro-5-(phosphonomethyl)[1,1′-biphenyl]-3-propanoicacid), SDZ 220-040((S)-a-Amino-2′,4′-dichloro-4-hydroxy-5-(phosphonomethyl)-[1,1′-biphenyl]-3-propanoicacid), CGP 37849 ((E)-(±)-2-Amino-4-methyl-5-phosphono-3-pentenoicacid), CGP 39551 ((E)-(±)-2-Amino-4-methyl-5-phosphono-3-pentenoic acidethyl ester); Glycine Site Antagonists such as D-cycloserine, CNQX(6-Cyano-7-nitroquinoxalme-2,3 dione), 7-Chlorokynurenic acid(7-Chloro-4-hydroxyquinoline-2-carboxylic acid), ACBC(1-Aminocyclobutane-1-carboxylic acid), 7-Chlorokynurenate,(S)-(−)-HA-966 ((S)-(−)-3-Ammo-1-hydroxypyrrolidin-2-one),5,7-Dichlorokynurenic acid (DCKA,5,7-Dichloro-4-hydroxyquinoline-2-carboxylic acid), L-701,252(7-Chloro-3-(cyclopropylcarbonyl)-4-hydroxy-2(1H)-quinolinone),L-689,560 (trans-2Carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline),Felbamate (2-Phenyl-1,3-propanedioldicarbamate), L-701,324(7-Chloro-4-hydroxy-3-(3-phenoxyl)phenyl-2(1H)-quinolinone), CGP 78608hydrochloride([(1S)-1-[[(7-Bromo-1,2,3,4-tetrahydro-2,3-dioxo-5-quinoxalinyl)methyl]amino]ethyl]phosphonicacid hydrochloride), Gavestinel (GV-150,526), Lacosamide,L-phenylalanine, 1-Aminocyclopropanecarboxylic acid (ACPC); Ion ChannelAntagonists such as (±)-1-(1,2-Diphenylethyl)piperidine maleate,Dizocilpine ((+)-MK 801 maleate),(5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-iminemaleate, (−)-MK 801 maleate((5R,10S)-(−)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cylcohepten-5,10-iminemaleate), Loperamide hydrochloride(4-(4-Chlorophenyl)-4-hydroxy-N,N-dimethyl-a,a-diphenyl-1-piperidinebutanamidehydrochloride), Remacemide hydrochloride(2-Amino-N-(1-methyl-1,2-diphenylethyl)acetamide hydrochloride), IEM1460(N,N,N,-Trimethyl-5-[(tricyclo[3.3.1.13,7]dec-1-ylmethyl)amino]-1-pentanaminiumbromidehydrobromide), ketamine, Norketamine hydrochloride(2-Amino-2-(2-chlorophenyl)cyclohexanone hydrochloride), N20Chydrochloride (2-[(3,3-Diphenylpropyl)amino]acetamide hydrochloride),dextromethorphan, AZD6765 (Lanicemine), Aptiganel (Cerestat, CNS-1102),HU-211, Rhynchophylline, Amantadine and related compounds,nitromemantine, Memantine (hydrochloride3,5-Dimethyl-tricyclo[3.3.1.13.7]decan-1-amine hydrochloride) andrimantidine and similar derivatives described in U.S. Pat. Nos.6,071,876, 5,801,203, 5,747,545, 5,614,560, 5,506,231, PCT Applications01/62706 and WO 01/48516, Dextrallorphan, Dextromethorphan (DXM) andanalogs thereof described in U.S. Pat Pub No: 20100137448, Dextrorphan,Diphenidine, Ethanol, Eticyclidine, Gacyclidine, Ibogaine, Magnesium,Methoxetamine, Nitrous oxide, Phencyclidine (PCP), Rolicyclidine,Tenocyclidine, Methoxydine(4-meo-pcp), Tiletamine, Xenon, Neramexane(1,3,3,5,5-pentamethylcyclohexanamine), Etoxadrol, Dexoxadrol, WMS-2539,NEFA, Delucemine, 8A-PDHQ; polyamine site antagonists such as spermine,N-(4-Hydroxyphenylacetyl)spermineN—(N-(4-Hydroxyphenylacetyl)-3-ammopropyl)-(N′-3-aminopropyl)-1,4-butanediamine,N-(4-Hydroxyphenylpropanoyl) spermine trihydrochloride(N—(N-(4Hydroxyphenylpropanoyl)-3-aminopropyl)-(N′-3-ammopropyl)-1,4-butanediaminetrihydrochloride), Arcaine sulfate (N,N′-1,4-Butanediylbisguanidinesulfate), Ifenprodil hemitartrate(2-(4-Benzylpiperidino)-1-(4-hydroxyphenyl)-1-propanol hemitartrate),Synthalin sulfate (N,N′-1,10-Decanediylbisguanidine sulfate), Eliprodil(a-(4-Chlorophenyl)-4-[(4-fluorophenyl)methyl]-1-piperidineethanol);other NMDA selective Antagonists such as the NR2B antagonists Ro 25-6981maleate((αR,βS)-α-(4-Hydroxyphenyl)-β-methyl-4-(ρhenylniethyl)-1-piperidinepropanolmaleate) (CP101,606 or Traxoprodil), Bilobalide, Lipocortin-1, CerebralFluid Zinc level elevators, riluzole, ifenprodil, iamotrigine,spermidines, flupirtine, levernopamil,1-phenyl-2-(2-pyridyl)ethanamine), agniatine; synthetic opioids such asMeperidine, Methadone, Dextropropoxyphene, Tramadol and Ketobemidone;compounds having NMDA-antagonist-like properties including hydrogenatedpyrido[4,3-b]indole, dimebon described in U.S. Pat Pub. No. U.S.2010/0178277 and Huperzine A.

In some embodiments, the NMDAR antagonist is selected from Amantadine,AZD6765, Dextrallorphan, Dextromethorphan, Dextrorphan, Diphenidine,Dizocilpine (MK-801), Ethanol, Eticyclidine, Gacyclidine, Ibogaine,Memantine, Methoxetamine, Nitrous oxide, Phencyclidine, Rolicyclidine,Tenocyclidine, Methoxydine, Tiletamine, Xenon, Neramexane, Eliprodil,Etoxadrol, Dexoxadrol, WMS-2539, NEFA, Remacemide, Delucemine, 8A-PDHQ,Aptiganel, HU-211, Remacemide, Rhynchophylline, Ketamine,1-Aminocyclopropanecarboxylic acid (ACPC), 7-Chlorokynurenate′ DCKA(5,7-dichlorokynurenic acid), Kynurenic acid, Lacosamide,L-phenylalanine, Neurotransmitters, Psychedelics, Long-termpotentiation, and NMDA. In particular embodiments, the NMDAR antagonistis selected from ketamine, remacemide, and D-cycloserine.

In other embodiments, the NMDAR antagonist can be a low-trapping NMDARchannel blocker. An example of a low-trapping NMDAR channel blocker isan arylalkyl-amine having the following formula:

-   -   wherein, Ar¹ and Ar², which may be the same or different,        independently represent phenyl or phenyl substituted by one or        more of amino, nitro, halogen, hydroxy, C₁-C₆ alkoxy, C₁-C₆        alkyl or cyano;    -   R¹ represents hydrogen, C₁-C₆ alkyl, C₁-C₆ alkoxycarbonyl;    -   R² represents hydrogen or COCH₂ NH₂;    -   R³ represents hydrogen or C₁-C₆ alkyl;    -   in addition, when R² represents hydrogen either one or both of        Ar¹ and Ar^(e) may also represent 2-, 3- or 4-pyridinyl and R¹        may also represent trihalomethyl;    -   or a pharmaceutically acceptable salt thereof.

Examples of NMDAR antagonists having the above noted formula include:

-   1,2-diphenylethylamine;-   1,2-diphenyl-2-propylamine;-   1,2-bis(4-fluorophenyl)-2-propylamine;-   1,2-diphenyl-2-butylamine;-   (−)1,2-diphenyl-2-propylamine;-   (+)1,2-diphenyl-2-propylamine;-   2,3-diphenyl-2-aminopropanoic acid methyl ester;-   N-methyl-1,2-diphenyl-2-propylamine;-   N-methyl-1,2-diphenylethylamine;-   1-(3-nitrophenyl)-2-phenyl-2-propylamine;-   1-(3-chlorophenyl)-2-phenyl-2-propylamine;-   1-(3-bromophenyl)-2-phenyl-2-propylamine;-   1-(3-cyanophenyl)-2-phenyl-2-propylamine;-   2-(2-methylphenyl)-1-phenyl-2-propylamine;-   1-(4-chlorophenyl)-2-phenyl-2-propylamine;-   1-phenyl-2-(3,4-dichlorophenyl)-2-propylamine;-   1-phenyl-2-(3-methoxyphenyl)-2-propylamine;-   1-(4-hydroxyphenyl)-2-phenyl-2-propylamine;-   1-(4-hydroxyphenyl)-2-phenylethylamine;-   1-phenyl-2-(4-hydroxyphenyl)ethylamine;-   1,2-bis(4-hydroxyphenyl)ethylamine;-   1-phenyl-2-(4-hydroxyphenyl)-2-propylamine;-   1,2-bis(4-hydroxyphenyl)-2-propylamine;-   1-(2-pyridinyl)-2-phenylethylamine;-   1-(3-pyridinyl)-2-phenylethylamine;-   1-(4-pyridinyl)-2-phenylethylamine;-   1-phenyl-2-(2-pyridinyl)ethylamine;-   1-phenyl-2-(3-pyridinyl)ethylamine;-   1-phenyl-2-(4-pyridinyl)ethylamine;-   N-methyl-1-(3-pyridinyl)-2-phenylethylamine;-   3,3,3-trifluoro-1,2-diphenyl-2-propylamine;-   N-methyl-3,3,3-trifluoro-1,2-diphenyl-2-propylamine;-   2-amino-N-(1,2-diphenyl-1-methylethyl)acetamide;-   2-amino-N-(1,2-diphenylethyl)acetamide; and-   2-amino-N-[1,2-bis(4-fluorophenyl)-1-methylethyl]acetamide.

The compounds described above are basic compounds and may be used assuch or pharmaceutically acceptable acid addition salts may be preparedby treatment with various inorganic or organic acids, such ashydrochloric, hydrobromic, sulfuric, phosphoric, acetic, lactic,succinic, fumaric, malic, maleic, tartaric, citric, benzoic,methanesulfonic or carbonic acids. Methods of making the compoundsdescribed above are disclosed, for example, in U.S. Pat. No. 5,605,916,which is incorporated herein by reference in its entirety.

In other embodiments, the NMDAR antagonist can be a low-trapping NMDARchannel blocker having the following formula:

-   -   where:    -   R¹ and R² are independently phenyl or 4-fluorophenyl;    -   R³ is hydrogen, C1-6 alkyl or methoxycarbonyl;    -   R⁴ is hydrogen or methyl; and pharmaceutically acceptable salts        thereof or an active metabolite thereof.

In still other embodiments, the low-trapping NMDAR channel blocker caninclude 2-amino-N-(1,2-diphenyl-1-methylethyl)acetamide (remacemide) ora pharmaceutically acceptable salt thereof (e.g., hydrochloride salt) orits active metabolite. Examples of active metabolites of remacemideinclude desglycinyl metabolites, such as FPL 12495 or ARL 12495AA, FPL14331, FP1 14465, FPL 15455, FPL 14991, FPL 14981, FPL 13592, and FPL15112. In some embodiments, the active metabolite can be FPL 12495 orARL 12495AA.

In some embodiments, the NMDAR antagonist can be administered incombination with a TrkB agonist (e.g., in a combination therapy) totreat a pervasive development disorder, such as RTT or autism spectrumdisorder. It was found that RTT mice exhibit increased activity,associated with synaptic hyperexcitability (measuredelectrophysiologically) in brainstem circuits critical for respiratoryand autonomic control. Hyperexcitability in brainstem respiratory andautonomic circuits is associated with deficits in Brain DerivedNeurotrophic Factor (BDNF) and reduced activation of its receptor, TrkB.Moreover, exogenous BDNF and the small molecule TrkB ligand, LM22A-4acutely reverse synaptic hyperexcitability in these circuits andeliminate apneic breathing in vivo.

The phrase “combination therapy” embraces the administration of a NMDARantagonists and a TrkB agonist as part of a specific treatment regimenintended to provide a beneficial effect from the co-action of thesetherapeutic agents. When administered as a combination, the NMDARantagonist and the TrkB agonist can be formulated as separatecompositions or in a single composition. Administration of thesetherapeutic agents in combination typically is carried out over adefined time period (usually minutes, hours, days or weeks dependingupon the combination selected).

When formulated as separate compositions, “combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, in a substantially simultaneous manner. Sequentialor substantially simultaneous administration of each therapeutic agentcan be effected by any appropriate route including, but not limited to,intraperitoneal routes, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intraperitoneal injection while theother therapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredby intraperitoneal injection or all therapeutic agents may beadministered orally. The sequence in which the therapeutic agents areadministered is not narrowly critical. “Combination therapy” also canembrace the administration of the therapeutic agents as described hereinin further combination with other biologically active ingredients, knowntherapeutic compounds and/or non-drug therapies (e.g., surgery).

A TrkB agonist for use in a combination therapy with the NMDARantagonist can include any agent capable of directly activating orindirectly promoting the activation of TrkB. For example, a TrkB agonistcapable of directly activating TrkB can include TrkB activatingantibodies (described in Qian et al., Novel agonist monoclonalantibodies activate TrkB receptors and demonstrate potent neurotrophicactivities. J Neurosci 2006; 26:9394-9403 and US2010/0297115). A TrkBagonist capable of directly activating TrkB can also include smallmolecules that function as direct and specific TrkB activating ligands,such as but not limited to 7,8-dihydroxyflavone (7,8-DHF) (described inJang et al., A selective TrkB agonist with potent neurotrophicactivities by 7,8-dihydroxyflavone. Proc Natl Acad Sci USA 2010;107:268) and the non-peptide BDNF loop 2 domain mimetic, LM22A-4(described in Massa et al., Small molecule BDNF mimetics activate TrkBsignaling and prevent neuronal degeneration in rodents. J Clin Invest.2010; 120(5):1774-1785).

In some aspects, the TrkB agonist is an agent that transactivates TrkBincluding but not limited to adenosine or an adenosine agonist such asCGS 21680 described in Lee and Chao, Activation of Trk neurotrophinreceptors in the absence of neurotrophins, Proc Natl Acad Sci USA. 2001Mar. 13; 98(6):3555-60).

Mature brain-derived neurotrophic factor (BDNF) is a secreted proteinthat, in humans, is encoded by the BDNF gene. BDNF acts via tworeceptors, the p75 neurotrophin receptor and the TrkB tyrosine kinasereceptor. Thus, in some embodiments, a TrkB agonist can include anyagent capable of increasing BDNF activation of TrkB in a subject. Forexample, such agents can include BDNF itself and known therapeuticcompounds that promote endogenous BDNF production in a subject includingbut not limited to ampakines, inhibitors of BDNF gene repression, mixedlineage kinase inhibitors, and antidepressants.

Examples of ampakines can be allosteric modulators of the AMPA-receptor.“α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid”, or “AMPA”, or“glutamatergic” receptors are molecules or complexes of moleculespresent in cells, particularly neurons, usually at their surfacemembrane, that recognize and bind to glutamate or AMPA. The binding ofAMPA or glutamate to an AMPA receptor normally gives rise to a series ofmolecular events or reactions that result in a biological response. Thebiological response may be the activation or potentiation of a nervousimpulse, changes in cellular secretion or metabolism, or causing cellsto undergo differentiation or movement. Allosteric modulators of theAMPA-receptor that can be used for practicing the methods describedherein and methods of making these compounds are disclosed in U.S. Pat.Nos. 5,488,049; 5,650,409; 5,736,543; 5,747,492; 5,773,434; 5,891,876;6,030,968; 6,274,600 B1; 6,329,368 B1; 6,943,159 B1; 7,026,475 B2 andU.S. Application 20020055508. The disclosures of these publications areincorporated herein by reference in their entireties, especially withrespect to the ampakines disclosed therein.

In some embodiments, the ampakine compound can include those compoundshaving the following general Formula I:

In this formula:

-   -   R¹ is a member selected from the group consisting of N and CH;    -   m is 0 or 1;    -   R² is a member selected from the group consisting of (CR⁸        ₂)_(n-m) and C_(n-m)R⁸ _(2(n-m)-2), in which n is 4, 5, 6, or 7,        the R⁸'s in any single compound being the same or different,        each R⁸ being a member selected from the group consisting of H        and C₁-C₆ alkyl, or one R⁸ being combined with either R³ or R⁷        to form a single bond linking the no. 3′ ring vertex to either        the no. 2 or the no. 6 ring vertices or a single divalent        linking moiety linking the no. 3′ ring vertex to either the no.        2 or the no. 6 ring vertices, the linking moiety being a member        selected from the group consisting of CH₂, CH₂CH₂, CH═CH, O, NH,        N(C₁-C₆ alkyl), N═CH, N═C(C₁-C₆ alkyl), C(O), O—C(O), C(O)—O,        CH(OH), NH—C(O), and N(C₁-C₆ alkyl)-C(O);    -   R³, when not combined with any R⁸, is a member selected from the        group consisting of H, C₁-C₆ alkyl, and C₁-C₆ alkoxy;    -   R⁴ is either combined with R⁵ or is a member selected from the        group consisting of H, OH, and C₁-C₆ alkoxy;    -   R⁵ is either combined with R⁴ or is a member selected from the        group consisting of H, OH, C₁-C₆ alkoxy, amino, mono(C₁-C₆        alkyl)amino, di(C₁-C₆ alkyl)amino, and CH₂OR⁹, in which R⁹ is a        member selected from the group consisting of H, C₁-C₆ alkyl, an        aromatic carbocyclic moiety, an aromatic heterocyclic moiety, an        aromatic carbocyclic alkyl moiety, an aromatic heterocyclic        alkyl moiety, and any such moiety substituted with one or more        members selected from the group consisting of C₁-C₃ alkyl, C₁-C₃        alkoxy, hydroxy, halo, amino, alkylamino, dialkylamino, and        methylenedioxy; R⁶ is either H or CH₂OR⁹;    -   R⁴ and R⁵ when combined form a member selected from the group        consisting of

-   -   in which: R¹⁰ is a member selected from the group consisting of        O, NH and N(C₁-C₆ alkyl);    -   R¹¹ is a member selected from the group consisting of O, NH and        N(C₁-C₆ alkyl);    -   R¹² is a member selected from the group consisting of H and        C₁-C₆ alkyl, and when two or more R¹²'s are present in a single        compound, such R¹²'s are the same or different;    -   p is 1, 2, or 3; and    -   q is 1 or 2; and    -   R⁷, when not combined with any R⁸, is a member selected from the        group consisting of H, C₁-C₆ alkyl, and C₁-C₆ alkoxy.

A further class of ampakine compounds is those of Formula II:

In Formula II:

-   -   R²¹ is either H, halo or CF₃;    -   R²² and R²³ either are both H or are combined to form a double        bond bridging the 3 and 4 ring vertices;    -   R²⁴ is either H, C₁-C₆ alkyl, C₅-C₇ cycloalkyl, C₅-C₇        cycloalkenyl, Ph, CH₂ Ph, CH₂ SCH₂ Ph, CH₂ X, CHX₂, CH₂ SCH₂        CF₃, CH₂ SCH₂ CH—CH₂, or

-   -   and R²⁵ is a member selected from the group consisting of H and        C₁-C₆ alkyl.

A particularly preferred compound is1-(Quinoxalin-6-ylcarbonyl)piperidine, having the following structure:

Another particularly preferred compound is1-(1,4-benzodioxan-6-ylcarbonyl)piperidine, having the followingstructure:

In another embodiment, the ampakine is a compound of formula III:

-   -   in which:    -   R¹ is oxygen or sulfur;    -   R² and R³ are independently selected from the group consisting        of —N═, —CR═, and —CX;    -   M is ═N or ═CR⁴—, wherein R⁴ and R⁸ are independently R or        together form a single linking moiety linking M to the ring        vertex 2′, the linking moiety being selected from the group        consisting of a single bond, —CR₂—, —CR═CR—, —C(O)—, —O—,        —S(O)_(y)—, —NR—, and —N═;    -   R⁵ and R⁷ are independently selected from the group consisting        of —(C₂)—, —C(O)—, —CR═CR—, —CR═CX—, —C(RX)—, —CX₂—, —S—, and        —O—; and    -   R⁶ is selected from the group consisting of —(CR₂)—, —, —C(O)—,        —CR═CR—, —C(RX)—, —CR₂—, —S—, and —O—;    -   Wherein    -   X is —Br, —Cl, —F, —CN, —NO₂, —OR, —SR, —NR₂, —C(O)R—, —CO₂ R,        or —CONR₂; and    -   R is hydrogen, C₁-C₆ branched or unbranched alkyl, which may be        unsubstituted or substituted with one or more functionalities        defined above as X, or aryl, which may be unsubstituted or        substituted with one or more functionalities defined above as X;    -   m and p are independently 0 or 1;    -   n and y are independently 0, 1 or 2.

The ampakine compounds described above for use in the methods describedherein can be prepared by conventional methods known to those skilled inthe art of synthetic organic chemistry as described in US Pat Pub No:20100035877A1.

Additional agents capable of increasing BDNF activation of TrkB in asubject include AMPA selective compounds that enhance the stimulation ofAMPA receptors and BDNF expression in the brain stem. Exemplary AMPAselective compounds for use herein include but are not limited tobenzoxazines, such as the pyrolidine derivative racetam drugs piracetamand aniracetam, the CX-series of drugs which encompass a range ofbenzoylpiperidine and benzoylpyrrolidine structures, such as CX-546,CX-614, CX-691, CX-717, Org 26576, CX-701, CX-1739, CX-1763 and CX-1837,benzothiazide derivatives such as cyclothiazide and IDRA-21,biarylpropylsulfonamides such as LY-392,098, LY-404,187, LY-451,646 andLY-503,430, 4-(benzofuran-5-yl carbonyl)morpholine, substitutedbenzotriazinone and substituted benzopyrimidione described inUS2010/02067728, bicyclic amides described in U.S. Pat. No. 8,119,632,and 3-substituted benzo-1,2,3]-triazin-4-one compounds described inUS2010/0041647.

In addition to BDNF and the transporter and receptor NMDA, loss offunction mutations of MeCP2 alters expression and activity of theneurotransmitter gamma-amino butyric acid GABA. Reduced GABAergicsignaling resulting from MeCP2 loss has significant effects on synaptictransmission in several brain regions important for respiratory controlincluding the ventrolateral medulla. It has been previously observedthat a reduction in inhibitory postsynaptic currents (IPSCs) in theventrolateral medulla of Mecp2-null mice resulted from reduced levels ofGABA and decreased expression of GABA receptor (GABAR). Therefore,another embodiment relates to a combination therapy for treating Rettsyndrome including administering to a subject in need thereof an NMDARantagonist, a TrkB agonist and a GABAR agonist.

A GABAR agonist can include any agent that acts to directly orindirectly stimulate or increase the effect of the GABA receptor.Exemplary GABAR agonists can include but are not limited to GABAanalogs, such as Neurontin (Gabapentin), PD-0200, 390 (atagabalin) andLyrica (pregabalin).

GABAR agonists can also include gamma-amino butyric acid A (GABA-A)agonists, gamma-amino butyric acid B (GABA-B) agonists and/orcombinations thereof. Exemplary GABA-A agonists for use in the presentinvention can be selected from the benzodiazepine groups acamprosate,barbiturates, ethanol, methaqualone, muscimol, nonbenzodiazepines(zaleplon, zolpidem, zopiclone), picamilon, progabide, and tiagabine.Exemplary GABA-B agonists can be selected from4-Amino-3-(4-chlorophenyl)butanoic acid ((RS)-Baclofen),(R)-4-Amino-3-(4-chlorophenyl)butanoic acid ((R)-Baclofen), CGP35024,CGP44532, 3-Aminopropyl(methyl)phosphinic acid (SKF 97541),1,4-Butanediol, GBL (γ-Butyrolactone), GHB (γ-Hydroxybutyric acid), GHV(γ-Hydroxyvaleric acid), GVL (γ-Valerolactone), lesogaberan, andphenibut.

The therapeutic agents described herein can be provided inpharmaceutical compositions for administration to a subject for thetreatment of a pervasive develop disorder, such as Rett syndrome orautism spectrum disorder. In some embodiments, a pharmaceuticalcomposition can include a therapeutically effective amount of an NMDARantagonist alone and or in combination with a TrkB agonist and/or aGABAR agonist and a pharmaceutically acceptable diluent or carrier. Acombination therapy described herein can include the amount of acombination of therapeutic agents described herein effective toameliorate biochemical and functional abnormalities associated withloss-of-function mutations of the gene encoding methyl-CpG bindingprotein 2 (MeCP2) in a subject.

In some embodiments, a therapeutically effective amount of an NMDARantagonist can be the amount required to significantly improve at leastone measure of forebrain function in a subject having Rett syndrome. Atherapeutically effective amount of an NMDAR antagonist can be theamount required to reverse hypoactivity in forebrain circuits ofsubjects having Rett syndrome. In some embodiments, a therapeuticallyeffective amount of an NMDAR antagonist can be the amount of an NMDARantagonist required to reverse abnormal synaptic phenotypes in thecortex of subjects having Rett syndrome such as deficits in mTORsignaling, decreased density of dendritic spines and/or abnormal E/Ibalance.

In some embodiments, a therapeutically effective amount of a TrkBagonist can be the amount of a TrkB agonist required to measurablyincrease BDNF expression in the brainstem of a subject, the amount of aTrkB agonist required to measurably increase levels of TrkBphosphorylation in the brainstem of a subject, the amount required toacutely reverse synaptic hyperexcitability in brainstem respiratory andautonomic neural circuits in the subject, and/or the amount required toimprove respiratory function in the subject, e.g., eliminate apneicbreathing. In an exemplary embodiment, the administration of 50 mg/kg ofLM22A-4 B.I.D for 4 weeks rescued wild-type levels of TrkBphosphorylation in the medulla and pons and restored wild-type breathingfrequency in a subject.

In some embodiments, a therapeutically effective amount of a GABARagonist can be the amount of a GABAR agonist required to measurablyincrease GABAergic signaling in a subject and/or the amount required tomeasurably increase synaptic transmission in brain regions important forrespiratory control such as the ventrolateral medulla of a subject.

The therapeutic agents described herein are capable of further formingboth pharmaceutically acceptable acid addition and/or base salts. All ofthese forms can be administered to the subject as part of apharmaceutical composition for the treatment of a persuasive developmentdisorder, such as Rett syndrome.

For preparing pharmaceutical compositions from the therapeutic agents ofthe present invention, pharmaceutically acceptable carriers can be inany suitable form (e.g., solids, liquids, gels, aerosols, etc.). Solidform preparations include, but are not limited to, powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. The presentinvention contemplates a variety of techniques for administration of thetherapeutic compositions. Suitable routes include, but are not limitedto, oral, rectal, transdermal, vaginal, transmucosal, or intestinaladministration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections, among others. Indeed, it is not intended thatthe present invention be limited to any particular administration route.

For injections, the agents may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological saline buffer. For suchtransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

In powders, the carrier is a finely divided solid which is in a mixturewith the finely dived active component. In tablets, the active componentis mixed with the carrier having the necessary binding properties insuitable proportions, which has been shaped into the size and shapedesired.

The powders and tablets can contain from five or ten to about seventypercent of the active compounds. Suitable carriers include, but are notlimited to, magnesium carbonate, magnesium stearate, talc, sugar,lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose,sodium carboxymethylcellulose, a low melting wax, cocoa butter and thelike, among other embodiments (e.g., solid, gel, and liquid forms). Theterm “preparation” is intended to also encompass the formation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

For preparing suppositories, in some embodiments of the presentinvention, a low melting wax, such as a mixture of fatty acid glyceridesor cocoa butter; is first melted and the active compound is dispersedhomogeneously therein, as by stirring. The molten homogenous mixture isthen poured into convenient sized molds, allowed to cool, and thereby tosolidify in a form suitable for administration

Liquid form preparations include, but are not limited to, solutions,suspensions, and emulsions (e.g., water or water propylene glycolsolutions). For parenteral injection, in some embodiments of the presentinvention, liquid preparations are formulated in solution in aqueouspolyethylene glycol solution. Aqueous solutions suitable for oral usecan be prepared by dissolving the active component in water and addingsuitable colorants, flavors, and stabilizing and thickening agents, asdesired.

Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

General procedures for preparing pharmaceutical compositions aredescribed in Remington's Pharmaceutical Sciences, E. W. Martin, MackPublishing Co., PA (1990), which is herein incorporated by reference init entirety.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg/kg per day to about 100 mg/kg per day,for example, ranging from 10 mg/kg per day to about 50 mg/kg per dayaccording to the particular application and the potency of the activecomponent. The composition can, if desired, also contain othercompatible therapeutic agents.

In particular embodiments, the quantity of an NMDAR antagonist in a unitdose preparation can be a sub-anesthetic dose. For example, the quantityof Ketamine in a unit dose can be a sub-anesthetic dose ranging fromabout 1 mg/kg to 20 mg/kg per day. In a particular embodiment, thequantity of Ketamine in a unit dose can be about 8 mg/kg per day. Inother embodiments the quantity of the NMDAR antagonist Remacemide in aunit dose can range from about 3 mg/kg to 120 mg/kg per day. In anotherexemplary embodiment, the quantity of a TrkB agonist LM22A-4 in a unitdose can range from about 50 mg/kg to 150 mg/kg per day.

The assessment of the clinical features and the design of an appropriatetherapeutic regimen for the individual patient is ultimately theresponsibility of the prescribing physician. It is contemplated that, aspart of their patient evaluations, the attending physicians know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physiciansalso know to adjust treatment to higher levels, in circumstances wherethe clinical response is inadequate, while precluding toxicity. Themagnitude of an administrated dose in the management of the disorder ofinterest will vary with the severity of the condition to be treated, thepatient's individual physiology, biochemistry, etc., and to the route ofadministration. The severity of the condition, may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and dose frequency will also vary according to the age, bodyweight, sex and response of the individual patient.

The following examples are included to demonstrate different embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples, which follow representtechniques discovered by the inventor to function well in the practiceof the claimed embodiments, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the claims. All patents, patent applications, and publicationcited herein are incorporated by reference in their entirety.

EXAMPLES

We discovered that NMDA receptor (NMDAR) targeted therapies have utilityfor symptom reversal in RTT. In direct support of the therapeuticpotential of NMDAR antagonists for RTT, we showed that acute treatmentof Mecp2 mutant mice with a sub-anesthetic dose of ketamine (8 mg/kg), anon-competitive NMDAR antagonist, completely reverses a subset of mutantendophenotypes, including hypoactivity in forebrain circuits andabnormal sensorimotor gating. These results are consistent with priorevidence that ketamine acutely increases forebrain network activity bydisinhibiting cortical pyramidal cells. However, in addition to itsacute effects on cortical network activity, ketamine also rapidlystimulates dendritic growth, BDNF translation and expression of keysynaptic proteins through activation of mTOR signaling, which isdeficient in Mecp2 mutants (FIG. 3).

These findings provide evidence that ketamine can effect long-termsynaptic repair in RTT by enhancing structural and functionalconnectivity, as recently demonstrated in animal models of depressionand stress. In fact, the antidepressant actions of low-dose ketamine arenow attributed, at least in part, to these mTOR dependent effects onsynaptic structure and function. We use mTOR activation, synapticprotein expression and synapse structure and function, in addition tobehavior, as endpoints for measuring treatment efficacy in Mecp2 mutantmice.

Our finding that low-dose ketamine is effective at rescuing someabnormal phenotypes in Mecp2 mutants suggests that ketamine may be aneffective RTT therapeutic.

Example 1 Brain Activity Mapping in Mecp2 Mutant Mice Reveals FunctionalDeficits in Forebrain Circuits, Including Key Nodes in the Default ModeNetwork, that are Reversed with Ketamine Treatment

In this example, we used Fos mapping, combined with electrophysiology,to compare activity patterns across the brain in wild-type (Wt) andMecp2 mutant mice. Our data indicate marked and reproducible effects ofthe Mecp2 Null genotype on activity levels in different brain regions,including hyperexcitability in autonomic reflex pathways in thebrainstem and hypoexcitability in key nodes of the default mode networkin the forebrain. However, forebrain hypofunction can be reversed bytreatment with a sub-psychotomimetic dose of ketamine, which alsorescues behavioral dysfunction.

Materials and Methods

Animals: Mecp2^(tm1.1Jae) mice were purchased from the Mutant MouseRegional Resource Center (University of California Davis, Davis Calif.)and maintained on a mixed genetic background (129Sv, C57BL/6, BALB/c) bycrossing Mecp2^(tm1.1Jae) heterozygous females (Mecp2^(−/+), Het) withMecp2^(tm1.1Jae) Wt males (Mecp2^(+/y)).

Immunohistochemistry

Tissue Preparation

Three- and 6-week-old male mice, or 11-week-old female mice, were deeplyanesthetized by inhalation of isoflurane and perfused transcardiallywith PBS followed by ice-cold 4% paraformaldehyde in 0.1M phosphatebuffer, pH7.4, within 10 min. The kinetics of Fos protein induction anddegradation are such that potential changes resulting from anesthesiawould not be detectable within this timeframe. Brains were postfixed in4% paraformaldehyde for 2.5 h, cryoprotected in 25% sucrose overnight,then frozen in 2-methylbutane at −45° C. and stored at −80° C. Coronalsections were cut at 40 μm with a cryostat microtome (Jung Frigocut 2800N) and stored in PBS at 4° C.

Immunostaining

Free-floating 40 μm sections were processed for Fos immunostaining byblocking with 10% goat serum in dilution buffer (PBS, BSA, 0.3% TritonX-100) for 1.5 h and then incubated overnight at room temperature (20±2h) in rabbit polyclonal anti-c-Fos primary antibody (1:3000, Calbiochem)in dilution buffer plus 10% goat serum. After sequential rinse steps indilution buffer and PBS, sections were incubated in biotinylated goatanti-rabbit IgG secondary antibody (1:400, Vector Labs) in dilutionbuffer plus 15% goat serum for 1 h. After rinsing in PBS, sections wereincubated in avidin and biotinylated horseradish peroxidase complex(ABC, 1:150, Vector Labs). Finally, sections were developed using theSigma Fast diaminobenzidine and urea hydrogen peroxide set, mounted onSuper Frost Plus slides and coverslipped using VectaShield (VectorLabs). Specificity of Fos immunolabeling was verified by demonstratingthat preabsorption of the anti-c-Fos primary antibody with Fos peptide(Calbiochem) eliminated nuclear staining. To determine whetherFos-positive cells were neurons or astrocytes, a subset of sections weredouble stained with anti-c-Fos and either mouse anti-MAP2 (1:1000,Sigma) or mouse anti-GFAP (1:1000, Calbiochem), respectively. Regardlessof genotype (Wt vs. Null), we only observed Fos labeling in neuronsexpressing the neuronal cytoskeletal protein MAP2 and saw nocolocalization with the glial protein marker GFAP (glial fibrillaryacidic protein) (n=2 animals per genotype). In addition, a subset ofsections were double stained for Fos and either CaMKII (mouseanti-CaMKII, 1:10,000, Abcam), a marker for glutamatergic neurons, orparvalbumin (mouse anti-parvalbumin, 1:1500, Millipore), which isexpressed by a subpopulation of GABAergic neurons. Alexa Fluor488-conjugated goat anti-mouse secondary antibody (1:1000, or 1:2000 forCaMKII, Invitrogen) was used with each double stain.

Ketamine Injections

Animals were administered ketamine (8, 20, or 100 mg/kg, i.p.) or anequivalent volume of saline and then returned to their home cage for 90min. Subsequently, the animals were deeply anesthetized by inhalation ofisoflurane, perfused transcardially, and processed for Fos staining asdescribed above.

Data Analysis

Sections were visualized and photographed using an AxioSkop2 microscope(Zeiss) equipped with a Quantifire XI microscope camera (Optronics). Fospositive cells were counted using point by-point analysis withNeurolucida software (MBF Bioscience). Before analysis, thephotomicrographs were coded so that the observers were blinded to thegenotype. Cells were counted in every third section through the nTS, andin a representative subset of sections through the other brain regionsanalyzed. All sections were analyzed twice by two independent andblinded observers, and respective counts were averaged. The nTS wassampled at 12 levels through the rostro-caudal extent of the nucleus andthe average counts at each level were then added together to estimatethe total number of labeled cells per animal. To define genotype effectson Fos expression, G*Power 3 power analysis software was used todetermine group sizes.

Electrophysiology Slice Preparation

Horizontal brainstem slices were prepared from 3- and 5- to 7-week-oldMecp2 Null and Wt male mice Animals were deeply anesthetized byinhalation of isoflurane and then decapitated. Brains were removed fromthe skull and placed in low Ca2+, ice-cold artificial CSF (ACSF)containing the following (in mM): 125 NaCl, 3 KCl, 1.2 NaH₂PO₄, 1 CaCl₂,1.2 MgSO4, 2 MgCl₂, 25 NaHCO₃, 10 D-glucose, and 0.4 L-ascorbic acid,equilibrated to pH 7.4 with 95% O₂/5% CO₂, for 2-5 min. Then, brainstemswere dissected, glued on the mounting platform of a vibratome (Leica, VT1000S), and horizontal sections containing the nucleus tractussolitarius (nTS), including a long segment of the tractus solitarius(TS) were cut at 220-250 μm. Slices were then transferred to recordingACSF (containing in mM: 125 NaCl, 3 KCl, 1.2 NaH₂PO₄, 2 CaCl₂, 1.2MgSO₄, 25 NaHCO₃, 10 D-glucose, and 0.4 L-ascorbic acid, equilibrated topH 7.4 with 95% O₂/5% CO₂) at −32° C. and allowed to recover from theprocedure for at least 30 min before recordings.

Recordings

Slices were placed into the recording chamber, held in place withnylon-wired grid and superfused with recording ACSF at 30-32° C. at aflow rate of 4-5 ml/min. For stimulation of presynaptic inputs to nTSneurons, a concentric bipolar stimulation electrode (Frederick Haer) wasplaced on the TS, the medullary tract containing the central axons ofcardiorespiratory and other primary afferent inputs to the brainstem,rostral to recording sites. Patch pipettes were pulled from thick-walledborosilicate glass capillaries, and filled with intracellular solution(containing in mM: 130 K_gluconate, 10 NaCl, 11 EGTA, 1 CaCl₂, 10 HEPES,1 MgCl₂, 2 MgATP, 0.2 NaGTP), had resistances between 4 and 7MΩ.Recordings were made within 2 regions of nTS at the level of, and caudalto the obex: (1) lateral to or within the TS, including theinterstitial, lateral and ventrolateral subnuclei [referred to aslateral nTS (lnTS)], (2) within the commissural subnucleus (nComm)Neurons were visualized with an upright Olympus microscope (BX51WIF).Cells were identified as second-order neurons if they receivedmonosynaptic input, defined as a low jitter of latency of evokedpostsynaptic responses (<250p s), at 0.5 Hz TS stimulation. Evoked,spontaneous and miniature EPSCs (eEPSCs, sPSCs, mEPSCs) were recordedfrom cells meeting this criterion in the whole-cell voltage-clampconfiguration at a holding potential of −60 mV. To record eEPSCs, the TSwas stimulated at 0.5 and 20 Hz. In a subset of nComm neurons,input-output curves were obtained by gradually increasing the stimulusintensity based on the neurons' individual threshold (thrsh, thrsh+10%,thrsh+50%, 2× thrsh, 5× thrsh). In all other experiments the stimulationintensity was set to threshold+10%, typically between 50 and 200 μA(stimulus duration 100 μs, 20 sweeps). To compare intrinsic neuronalexcitability between genotypes, action-potential properties and firingfrequency in response to current injection (50 pA increments) wererecorded in nComm neurons as well. Only neurons with a resting membranepotential of at least −40 mV upon breakthrough were accepted. Data wereacquired using pClamp software. Signals were amplified (Axopatch 200B,Molecular Devices), filtered at 2 kHz and digitized at 10 kHz.

Data Analysis

Spontaneous and evoked postsynaptic currents were analyzed with Clampfitand Microsoft Excel. In the analysis of eEPSCs, 20 sweeps were averagedwith Clampfit and the resulting eEPSC amplitudes were measured andcompared between genotypes at different stimulation intensities. ForsPSCs and mEPSCs, traces were digitally filtered at 1 kHz, events werecounted within 2 min segments, and instantaneous frequencies andamplitudes were analyzed. The detection threshold for sPSCs and mEPSCswas set as 1-1.5× peak-to-peak noise. Miniature EPSC rise time wasanalyzed as the time from onset to peak of individual events, and thedecay time was estimated as the time between peak and recross of thedetection threshold.

Prepulse Inhibition

Prepulse inhibition (PPI) of the acoustic startle response (ASR) wasmeasured to assess sensorimotor gating function using Med AssociatesStartle Response recording system. Mice were divided into four groups(Het ketamine, Het vehicle, Wt ketamine, Wt vehicle), and receivedinjections of either ketamine (8 mg/kg) or an equivalent amount ofsaline immediately before they were placed individually inside asmall-sized, nonrestrictive, cubical Plexiglas recording chamber 12.5inches (L)×2.5 inches (W)×1.75 inches (H)] fixed on an accelerometerplatform and allowed to acclimate for 5 min. Subsequently, the mouse wasexposed to 4 testing blocks. In the first testing block, the initialstartle response amplitude was determined by delivering a 40 ms pulse of120 dB broadband white noise and recording the maximum startle amplitude(Vmax). A baseline startle response was determined by repeating thisrecording paradigm for six consecutive trials (with 8-23 s between eachstimulus) and calculating the average Vmax measured in those trials. Inthe second and third testing blocks, the mice were exposed to a seriesof “pulse-only” or “prepulse-pulse pair” stimuli to determine the effectthat a reduced intensity prepulse had on the acoustic startle response.The pulse-only stimulus consisted of an 80 ms stimulus of 120 dB. Theprepulse-pulse pair trials were conducted by delivering a single 20 msprepulse, with an intensity of 73, 76 or 82 dB before an 80 ms stimuluswith an intensity of 120 dB. An average delay of 15 s (8-23 s) occurredbetween each stimulus. Each prepulse-pulse pair trial was repeated 10times, pulse only trials were repeated 12 times. The Vmax measured fromeach prepulse-pulse trial was compared with the Vmax measured from the120 dB pulse-only trials, and a percentage prepulse inhibition (% PPI)was calculated for each prepulse intensity. In the fourth testing block,startle response Vmax was recorded from 6 additional 40 ms pulses of 120dB broadband white noise (with 8-23 s between each stimulus) andcompared with the baseline startle response from the first testing blockto eliminate the possibility of habituation to the acoustic stimulusthroughout the test.

Statistical Analysis

All data are presented as means±SEM. Genotype-dependent differences wereanalyzed by unpaired two-tailed Student's t test. Multiple group datawere analyzed by one-way ANOVA with post hoc least significantdifference (LSD) test for intergroup comparisons. Miniature EPSCfrequency and amplitude distributions were compared with theKolmogorov-Smirnov test. Results were considered significant if the pvalue was <0.05.

Results

Fos Expression is Markedly Altered in the Mecp2 Null Brain Compared withWt Controls

Fos immunostaining has been widely used as a surrogate marker ofneuronal depolarization to map circuits and pathways in the normal brainthat are activated by specific types and patterns of neural stimulation.Given that excitatory-inhibitory imbalance has been documented withinvarious cell groups in the Mecp2 mutant brain, we hypothesized that Wtand Null animals would exhibit regional differences in Fos expressionand that these differences could be used to map sites of circuitdysfunction in the Null brain. To address this possibility, we initiallysurveyed Fos expression in serial sections throughout the rostro-caudalextent of the brain, from the olfactory bulbs to the spinomedullaryjunction, in Wt and Null mice at 3 and 6 weeks of age, i.e., before andafter the appearance of overt RTT-like symptoms. We found no obviouseffect of Mecp2 genotype on the number of Fos-positive neurons in3-week-old animals in any brain region examined (postnatal day 21±3;Null, N═4; Wt, n=4). However, there were marked and reproducibledifferences between Null and Wt animals at 6 weeks of age across theneuraxis (postnatal day 42±3; Null, n=5-7; Wt, n=5-7; Table 1).

Forebrain

The most dramatic effects of Mecp2 genotype on Fos expression wereobserved in cortical and subcortical limbic structures, including theprelimbic and infralimbic cortices, retrosplenial cortex, cingulatecortex, the nucleus accumbens (nAC, both core and shell), as well as thepiriform cortex, the motor cortex and lateral septal nuclei, all ofwhich showed significantly less Fos labeling in Null animals comparedwith Wt (FIG. 1; Table 1). A similar pattern was observed in theauditory, somatosensory, and primary visual cortices, as well as thecaudate/putamen. In cortical regions, genotype effects on Fos labelingdid not appear to exhibit any laminar specificity. Quantitative analysisrevealed that the number of Fos-positive cells in Nulls was reduced byup to 80% compared with Wt (% Wt; piriform cortex, 46.6%; nAC, 23.4%;cingulate cortex, 29.4%; retrosplenial cortex, 18.6%; prelimbic cortex,24.3%; infralimbic cortex, 26.3%; motor cortex, 15.6%; lateral septalnucleus, 32.9%; Table 1). No genotypic differences in Fos expressionwere noted in other forebrain regions, such as the hippocampus,including the dentate gyms and the CA1 and CA3 regions together (CA;Table 1) nor in the mammillary bodies, thalamus and hypothalamus.

Brainstem and Cerebellum

In the brainstem, the periaqueductal gray (PAG) and the nucleus of thesolitary tract (nTS), both of which are major cell groups involved inmodulation of autonomic homeostasis exhibited the strongest genotypedependent differences in Fos expression levels. Specifically, the NullPAG had significantly fewer Fos-positive cells compared with Wt (Wt,38.1±20.0; Null, 25.1±6.0; p<0.01; Table 1; FIG. 1F). This was dueprimarily to a deficit in the ventral subdivision (v1PAG, reduction of35.5%; Table 1), whereas Fos-expression was not significantly differentbetween Wt and Null in the lateral (WAG) and dorsal divisions (dPAG),despite strong trends toward lower expression in the Nulls (Table 1). Incontrast, Null mice exhibited significantly more Fos-positive cellsthroughout the rostrocaudal extent of the three major cardiorespiratorysubnuclei in nTS [medial (mnTS), commissural (nComm) and lateral nTS,including interstitial, lateral and ventrolateral subnuclei (lnTS)]compared with Wt (% Wt; mnTS; 306.5%; nComm; 245.0%, lnTS; 324.8%; FIG.2A-C; Table 1). However, no significant genotype-dependent effects werefound in the nucleus retroambiguus (nRA), the preBotzinger complex(pBC), pontine nucleus, or the cerebellum (Table 1).

TABLE 1 Quantification of Fos expression in selected brain regions inNull vs. Wt mice Male Wt Mall Null Medulla n = 7 n = 7 Medial nTS 106.5± 0.5   326.4 ± .09*** Lateral nTS 137.9 ± 0.7   447.9 ± 2.0***Commissural nTS 21.8 ± 0.2  53.4 ± 0.5* Nucleus retroambiguus 7.9 ± 1.211.9 ± 2.2^(#) preBotzinger Complex 23.6 ± 7.4  29.1 ± 4.7  Pons n = 5 n= 5 Pontine nucleus 322.7 ± 63.7  199.4 ± 51.5  Midbrain n = 5 n = 5Dorsal PAG 21.6 ± 32.8 13.9 ± 4.7  Lateral PAG 53.0 ± 25.2  35.9 ± 10.7*Ventral PAG 39.7 ± 12.3 25.6 ± 7.5* Forebrain n = 5 n = 5 Piriformcortex 266.3 ± 35.0  124.2 ± 19.7* Nucleus accumbens 114.4 ± 22.8   26.8± 7.9** Cingulate cortex  50.4 ± 13.08 14.8 ± 4.8* Retrosplenial cortex 39.7 ± 10.86  7.4 ± 1.6* Prelimbic cortex 34.6 ± 6.81  8.4 ± 3.6**Infralimbic cortex 26.6 ± 3.8   7.0 ± 2.2** Motor cortex 19.9 ± 3.8  3.1 ± 1.6** Lateral septal nucleus 48.6 ± 14.9 11.6 ± 4.9* Hippocampus(CA1 + CA3) 22.5 ± 8.3  14.0 ± 4.6  Hippocampus (DG) 14.3 ± 4.7  9.5 ±2.6 Data are displayed as mean ± SEM (*<0.05;**p < 0.01;***p < 0.001;^(#)p < 0.15)Genotype Effects on Fos Labeling are Associated with Altered SynapticExcitability

Although Fos has been widely validated as a marker of neural activity innormal animals, this has not previously been analyzed in animals lackingMeCP2. Therefore, to determine whether or not Mecp2 genotype effects onFos labeling indeed reflect differences in neural activity, patchclampelectrophysiological recordings were used to compare synaptic andneuronal excitability in Wt and Null mice at 5-7 weeks of age, using thenTS as a model system. The nTS is ideally suited for such analysesbecause of a clear anatomic segregation between presynaptic inputs inthe solitary tract (TS) and second-order neurons within the various nTSsubnuclei. Indeed, EPSC amplitudes evoked by TS stimulation at 0.5 Hz(20 sweeps; stimulation intensity at 10% above the neurons' individualthresholds) were significantly larger in Nulls compared with Wt in boththe lnTS and nComm (lnTS; Null, 314.1±29.3 pA, n=19; Wt, 198.1±22.7 pA,n=21; p<0.01, unpaired Student's t test; nComm; Null, 231.5±21.1 pA,n=40; Wt, 129.3±11.2 pA, n=31; p<0.001; FIG. 3A,B), consistent withprevious findings in the mnTS. To validate these genotypedependentdifferences and to facilitate comparisons across individual neurons andbetween genotypes, threshold-based input-output curves were recordedfrom a subset of nComm neurons at 0.5 Hz TS stimulation. Theserecordings revealed significantly higher eEPSC amplitudes in the NullnComm at the 3 intermediate stimulation intensities and strong trends atthe lowest and highest stimulation intensities (Wt, n=17; Null, n=16;FIG. 3E). Increasing TS-stimulation frequency to 20 Hz also revealedsignificantly larger eEPSC amplitudes in the Null lnTS, and a strongtrend in nComm as well (lnTS; Null, 251.8±35.7 pA; Wt, 162.6±23.2 pA,p<0.05; nComm; Null, 103.6±16.3 pA; Wt, 68.7±6.5 pA, p<0.052; FIG.3C,D). Frequency-dependent synaptic depression, a feature of primaryafferent synapses in nTS was unaffected by Mecp2 genotype in eithernComm or lnTS. Similarly, basic membrane properties, including membranepotential (Vm), membrane capacitance (Cm) and membrane resistance (Rm)were comparable between genotypes (Tables 2, 3). Likewise,action-potential properties were similar between the genotypes, andcurrent-induced step depolarization at −60 mV evoked comparable numbersof APs at all current levels except for 50 pA (the lowest level tested)which evoked fewer APs in Null cells compared with Wt (Tables 4, 5). Todefine potential genotype effects on spontaneous network activity innTS, sPSCs were recorded and analyzed in 2 min intervals. In the lnTS,we detected significantly more events in Nulls compared with Wt (Null,1165.3±169.3, n=16; Wt, 718.1±141.6, n=17, p<0.05) which resulted in asignificantly higher instantaneous frequency (Null, 29.0±3.1 Hz; Wt,18.9±2.4 Hz, p<0.05; FIG. 4A,B). Instantaneous sPSC frequency was alsoincreased in the Null nComm compared with Wt (Null, 28.4±2.3 Hz, n=36;Wt, 21.2±2.1 Hz, n=28, p<0.05; FIG. 4B), in association with a strongtrend toward an increase in the number of spontaneous events (Null,1231.9_160.8; Wt, 867.2±127.3; p<0.08). Addition of the AMPA-receptorblocker 6-cyano-7-nitroquinoxaline-2,3-dione to the superfusate (10 μM)completely abolished sPSCs and reduced eEPSC amplitudes by 92.2±1.4%(n=10), indicating that primary afferent transmission in nTS is mainlymediated by AMPA-receptors. To specifically examine how Mecp2 genotypemay affect spontaneous presynaptic release of excitatory transmitter,miniature EPSCs were recorded in the presence of bicuculline (10 μM) andTTX (0.5 μM) and analyzed in 2 min intervals. Since we found similargenotype effects in the nComm and lnTS, data from both subnuclei werepooled. In Nulls, both the number of events and the instantaneous mEPSCfrequency were significantly increased compared with Wt (Null,1006±162.6 events, 24.6±2.9 Hz, n=9; Wt, 461.6±94.0 events, 15.9±2.4 Hz,n=7; p values <0.05; FIG. 4C, D). Accordingly, the cumulative frequencydistribution curve showed a significant right shift in the Nulls(Kolmogorov-Smirnov test, p<0.05, FIG. 4D). Moreover, the cumulativemEPSC amplitude distribution curve also displayed a significant rightshift in the Nulls, indicating larger mEPSC amplitudes(Kolmogorov-Smirnov test, p<0.05, FIG. 4E). We did not observe genotypedifferences in mEPSC rise or decay times (rise time; Wt, 1.00±0.11 ms;Null, 0.91±0.09 ms; decay time; Wt, 2.91±0.49 ms; Null, 2.75±0.36 ms).

Elevated Fos Expression and Synaptic Hyperexcitability Develop inParallel in Mecp2 Nulls

Analysis of 6-week-old animals revealed a strong association betweenelevated Fos expression and synaptic hyperexcitability within specificsubnuclei in the Null nTS compared with Wt. To further explore how thesetwo endophenotypes may be linked to each other, and to the onset ofdisease, we quantified Fos levels and analyzed synaptic excitability inthe nTS of 3 week-old mice before the appearance of overt symptoms. Incontrast to 6-week-old animals, Fos was expressed at relatively lowlevels in the nTS of both Null and Wt animals at 3 weeks, and we saw nosignificant effect of genotype in any subnucleus of the nTS (Wt, n=4;Null, n=4; mnTS; Wt, 3.8±0.5 cells; Null, 3.5±0.4 cells; lnTS; Wt,4.4±0.7 cells; Null, 3.7±0.7 cells; nComm; Wt, 3.1±0.5 cells; Null,1.9±0.2 cells). To evaluate genotype effects on synaptic function in nTSat 3 weeks of age, we focused our analysis on the lnTS. With theexception of Vm, which was more negative in Wt neurons, there was noeffect of genotype on basic neuronal properties (Table 2).

TABLE 2 Membrane properties of second-order nTS relay neurons (InTS)Juvenile InTS Adult InTS Wt (n = 26) Null (n = 30) Wt (n = 21) Null (n =18) V_(m) −64.5 ± 2.1    −59.5 ± 1.4    −60.4 ± 1.8    −62.9 ± 2.6   (mV) C_(m) 37.8 ± 2.4  36.8 ± 2.7  34.6 ± 3.0  29.9 ± 1.9  (pF) R_(m)440.6 ± 60.0  462.0 ± 75.0  447.3 ± 56.3  464.5 ± 51.5  (MΩ) Data aredisplayed as mean ± SEM; *p < 0.05

Similarly, there was no effect of genotype on eEPSC amplitudes evoked byTS stimulation at 0.5 Hz (Wt, 322.8±38.2 pA, n=21; Null, 344.4±41.2 pA,n=27; FIG. 5A,B) or 20 Hz (Wt, 228.5±34.1 pA; Null, 215.9±33.3 pA; FIG.8A,B). Regardless of genotype, eEPSC amplitudes at 3 weeks of age werecomparable to those recorded in Nulls at 6 weeks (see above).Instantaneous sPSC frequency (Wt, 23.4±2.4 Hz, n=20; Null, 16.4_1.7 Hz,n_25, p<0.05) and number of events (Wt, 877.6±123.2; Null, 545.0±81.0,p<0.05) were significantly lower in juvenile Nulls compared withjuvenile Wt (FIG. 8C,D). To compare the spontaneous release ofexcitatory transmitter between the genotypes in presymptomatic mice inmore detail, we constructed mEPSC frequency and amplitude probabilitydistribution plots. In contrast to mEPSC analyses from 5- to 7-week-oldmice, both frequency and amplitude distribution plots showed asignificant left shift in the Nulls (Kolmogorov-Smirnov test, p<0.05;Wt, n=8; Null, n=9; FIG. 8E-G), indicating lower mEPSC frequency andsmaller mEPSC amplitudes. Miniature EPSC rise and decay times werecomparable between the genotypes (rise time; Wt, 0.86±0.08 ms; Null,0.79±0.09 ms; decay time; Wt, 1.96±0.15 ms; Null, 2.04±0.17 ms).

The NMDA Receptor Antagonist Ketamine Restores Wt Levels of FosExpression in the Null Forebrain

To determine whether or not decreased Fos expression in corticalstructures within the adult Null forebrain reflects an intrinsicinability to express Fos in the absence of MeCP2 or, alternatively,results from reduced network activity, we compared the effects ofketamine treatment in Null and Wt mice. Ketamine is an NMDA receptorantagonist that has previously been shown to upregulate Fos expressionin the limbic forebrain of mice and rats by disinhibiting corticalpyramidal cells. Indeed, acute treatment with ketamine (8 mg/kg, 20mg/kg, 100 mg/kg, i.p.) markedly increased Fos labeling within 90 min ofinjection in both Wt and Null animals compared with saline-injectedcontrols (n=3 for each group, FIG. 6). In both genotypes, Fos inductionwas strongest in the prelimbic, infralimbic, piriform, cingulate andretrosplenial cortices (FIG. 6). Quantitative analysis of Fos labelingin the piriform cortex revealed a dosedependent effect of ketamine onthe number of Fos-positive cells in the Nulls, including restoration ofWt levels at higher doses (Null vehicle, 41.3±9.2 cells; Null 8 mg/kgketamine, 82.9±_13.4 cells; Null 20 mg/kg ketamine, 83.2±18.2 cells;Null 100 mg/kg ketamine, 116.3±26.4 cells; Wt vehicle, 99.5±13.3 cells;Wt 100 mg/kg ketamine, 119.3±17.2 cells; FIG. 6C). These data indicatethat although Fos is downregulated in limbic forebrain structures in theabsence of MeCP2, Fos expression remains plastic and subject toinduction by factors that alter forebrain network activity.

Ketamine Rescues Abnormal PPI of Acoustic Startle in Mecp2 Hets

PPI of the ASR is a measure of sensorimotor gating and is widely used asan index of cognitive function in neuropsychiatric disorders, includingASDs. PPI measures the ability of a weak sensory input to modulatebehavioral responses to a subsequent strong sensory stimulus and therebyreflects the function of inhibitory circuitry thought to be critical fornormal cognition. Because the circuitry underlying PPI includesstructures that exhibit reduced Fos staining in Nulls, such as the mPFCand nAC and because ketamine treatment of Nulls rescues Fos expressionin these regions, we decided to use PPI as an index of forebrain circuitfunction in the absence and presence of ketamine. Heterozygous femaleMecp2 mutants (Hets) were used for these experiments because acousticstartle measurements can be unreliable in Nulls due to their relativelysmall size. Although, as described by others, overall levels of Fosexpression are lower in females compared with males, Fos wassignificantly reduced in the Het forebrain compared with Wt, as in maleNulls (Table 3). PPI was compared in vehicle- and drug-treated Hets andage and sex-matched Wt animals, using a sub-psychotomimetic dose ofketamine (8 mg/kg; n=9 for Wt vehicle and ketamine, n=8 for Het vehicleand ketamine). Vehicle-treated Hets exhibited a significant increase inPPI amplitude compared with vehicle treated Wt at all levels of prepulsetested (% PPI 73 dB; Wt, 11.5±5.3, Het, 37.6±4.1, p<0.001; % PPI 76 dB;Wt, 11.3±5.2, Het, 29.9±8.6, p<0.05; % PPI 82 dB; Wt, 20.9±3.2, Het,42.6±7.1, P<0.05; FIG. 7). Acoustic startle by itself was not differentamong groups (Startle amplitude; Wtvehicle, 974.8_109.8; Het vehicle,759.6±73.7). Acute treatment with ketamine restored PPI in Hets to Wtlevels (Het ketamine, % PPI 73 dB, 5.3±5.8; % PPI 76 dB, 17.8±2.9; % PPI82 dB, 21.4±6.4, FIG. 7), whereas ketamine treatment did not alter PPIin Wt (Wt ketamine, % PPI 73 dB, 7.8±4.1; % PPI 76 dB, 12.8±4.8; % PPI82 dB, 24.1±5.8; FIG. 10) and had no effect on acoustic startle alone(Wt ketamine, 798.6±106.3; Het ketamine, 862.7±149.5).

TABLE 3 Quantification of Fos expression in selected brain regions offemale Wt vs. Het mice Forebrain Female Wt (n = 11) Female Het (n = 9)Piriform complex 41.4 ± 3.7  31.8 ± 5.6^(# ) Nucleus accumbens 48.8 ±3.4   25.5 ± 5.6** Cingultate cortex 28.3 ± 6.6  26.1 ± 5.7 Retrosplenial cortex 7.4 ± 1.9  1.4 ± 0.3* Prelimbic cortex 28.5 ± 6.3 10.3 ± 2.9* Infralimbic cortex 21.8 ± 5.5  10.8 ± 2.3* Data aredisplayed as mean ± SEM (*p < 0.05; **p < 0.01; ^(#)p < 0.15)

Our findings demonstrate marked effects of Mecp2 genotype on expressionof the activity-dependent, immediate-early gene product Fos withinspecific forebrain and hindbrain networks, including many previouslyunrecognized sites of circuit dysfunction within the Mecp2 mutant brain.In view of the close spatial and temporal association between genotypeeffects on neural activity and Fos expression, our data indicate thatloss of MeCP2 results in a stereotyped pattern of activity changeswithin a defined subset of functionally interrelated brain circuits thatemerges during late postnatal development, coincident with theappearance of overt symptoms (Table 1).

Forebrain Circuitry and the Default Mode Network

Reduced expression of Fos in forebrain cortices is consistent withreports of hypoconnectivity in layer 5 cortical circuits in Mecp2mutants. However, a particularly striking feature of the Fos map in Nullmice is the marked reduction in labeling throughout the midline limbicnetwork, including the medial prefrontal (mPFC), cingulate andretrosplenial cortices compared with Wt. This pattern of hypoactivity issignificant because (1) these cortices are key nodes in the default modenetwork, a forebrain meta-circuit that also exhibits hypoactivity and/orreduced connectivity in human autism, and (2) the midline limbiccortices play a critical role in behavioral state regulation ofautonomic homeostasis, which is abnormal in RTT.

Ketamine Rescue of Mutant Fos and PPI Phenotypes

Our finding that forebrain deficits in Fos expression in Nulls can berescued by acute treatment with ketamine, even at subpsychotomimeticdoses, illustrates that reduced Fos labeling reflects a reversibledeficit in network activity, rather than an intrinsic inability toexpress Fos.

Circuits for Autonomic Homeostasis

Consistent with the pathophysiology of RTT, our data indicate circuitdysfunction in structures involved in both reflex and behavioral controlof cardiorespiratory function, including the medulla (nTS), midbrain(PAG) and forebrain limbic cortices in Nulls.

Example 2

This Example evaluated the present study was undertaken to evaluate theability of a small molecule, nonpeptide BDNF loop 2 domain mimetic,LM22A-4, which functions as a direct and specific partial agonist ofTrkB, but not p75, to increase TrkB activation and improve breathing ina mouse model of RTT. LM22A-4 was developed by in silico screening formimetics of BDNF loop domains that selectively activate TrkB in vitroand in vivo and promote recovery of motor function in a rodent model ofbrain trauma. Using heterozygous female Mecp2 mutant mice, we show thatdaily treatment with LM22A-4 is well tolerated and rescues wild-typelevels of TrkB phosphorylation and wild-type breathing frequency.

Materials and Methods

Animals

Mecp2tm1.1Jae mice were purchased and maintained on a mixed background(129Sv, C57BL/6, BALB/c) by crossing Mecp2^(tm1,1Jae) heterozygousfemales (Mecp2^(−/+), Het) with Mecp2^(tm1.1Jae) wild-type males(Mecp2⁺/^(y)). Experimental procedures were approved by theInstitutional Animal Care and Use Committee at Case Western ReserveUniversity.

BDNF Protein Measurements

BDNF concentrations were quantified by ELISA using the BDNF EmaxImmunoassay System (Promega) according to the manufacturer's protocol.The sensitivity of this BDNF ELISA assay is ˜1-3 pg BDNF/ml. Braintissues and nodose ganglia were rapidly dissected and quick-frozen ondry-ice. Brain tissue samples were homogenized in 200 μl of RIPA buffer(containing, in mM: 50 Tris-HCl, 1% Nonidet P-40, 0.25% sodiumdeoxycholate, 150 NaCl, 1 EDTA, pH 7.4) containing a mixture of proteaseinhibitors (Roche), and centrifuged at 16,000×g for 15 min at 4° C.Nodose ganglia were homogenized in 100 μl of the same buffer andcentrifuged at 14,000×g for 30 s at room temperature. The supernatantfrom each sample was collected and stored at −80° C. until further use.For brain tissue samples, an aliquot of the supernatant was used todetermine total protein content using the Bradford technique and 300 μgof total protein were used for BDNF ELISA. For BDNF ELISA, the entiresupernatant from each individual nodose ganglion was loaded.

TrkB, AKT, and ERK Phosphorylation Assays

The ratios of (1) phospho-TrkB^(Y817)/TrkB (full-length),phospho-ERK/ERK and phospho-AKT/AKT, (2) phospho-TrkB^(Y817)/actin,phospho-ERK/actin and phospho-AKT/actin, (3) TrkB (full-length)/actin,ERK/actin and AKT/actin and (4) TrkB (truncated)/actin were measured byWestern blot using the ECL Chemiluminescence System (GE Healthcare).Site-specific rabbit monoclonal antiphospho-TrkB^(Y817) antibody wasobtained from Epitomics and rabbit polyclonal TrkB antibody was obtainedfrom Millipore. Rabbit polyclonal ERK and AKT, mouse monoclonalphospho-ERK and phospho-AKT antibodies were obtained from Cell SignalingTechnology. Tissues were homogenized in a lysis buffer (containing, inmM: 20 Tris, pH 8.0, 137 NaCl, 1% Igepal CA-630, 10% glycerol, 1 PMSF,10 μg/ml aprotinin, 1 μg/ml leupeptin, 500 μM orthovanadate). Lysateswere centrifuged at 14,000×g for 10 min, then the supernatant wascollected and protein concentration was determined using the BCA ProteinAssay Reagent (Pierce).

Spectrometric Analysis of LM22A-4 Levels in the Brain

The brain concentration of LM22A-4 (custom synthesized by RicercaBiosciences, LLC) was evaluated in Het mice 1 h after a 50 mg/kgintraperitoneal dose using reverse-phase liquid chromatography withtriple-quadrupole tandem mass spectrometric (LC-MS/MS) detection.Atenolol, a drug that does not cross the blood-brain barrier, wasadministered orally as a control to correct for contamination by bloodpresent in the brain vascular space. Brainstem and forebrain sampleswere homogenized with a Virsonic 100 ultrasonic homogenizer and preparedfor analysis using acetonitrile precipitation by combining one volume ofsample with three volumes of acetonitrile. Samples were centrifuged andthe resulting supernatant was sampled for analysis using a CTC Leap PALautosampler (Leap Technologies) and two PerkinElmer series 200 micropumps. Chromatography was performed at ambient temperature using a 50×20 mm inner diameter, 4 μm Synergi Polar-RP analytical column(Phenomenex). The aqueous mobile phase (A) was 4 mm ammonium formate, pH3.5 and the organic mobile phase (B) was 10:90 (v/v) 4 mm ammoniumformate, pH 3.5/acetonitrile. The analyte was eluted with a gradientwhich changed linearly from 0 to 100% B in 3 min at a flow rate of 300μl/min. The total run time was 4.5 min and the injection volume was 10μl. The analyte was detected on a Sciex API 3000 triple-quadrupole massspectrometer equipped with a TurbolonSpray interface in the positiveelectrospray ionization mode (Applied Biosystems/MDS). The multiplereaction monitoring transitions and instrument settings were optimizedfor LM22A-4. Equipment operation, data acquisition, and data integrationwere performed using Analyst version 1.4.2. software (AppliedBiosystems). The drug injections, tissue extraction and LCMS/MS analysiswere performed by Absorption Systems.

Respiratory Function

Respiratory patterns were analyzed at 8, 10, and 12 weeks after birth inunrestrained mice using whole-body plethysmography as describedpreviously. Measurements were taken from quiet breathing periods of atleast 5 min total duration, and apneas were defined as pauses inbreathing greater than two times the average breath duration calculatedfor each animal. LM22A-4 treatment. Wt and Het littermates were dividedinto 4 groups: Wt vehicle-treated (100 μl of 0.9% NaCl, i.p., b.i.d.),Wt drug treated (50 mg/kg LM22A-4 in 0.9% NaCl, i.p., b.i.d.), Hetvehicle-treated (100 μl of 0.9% NaCl, i.p., b.i.d.), and Hetdrug-treated (50 mg/kg LM22A-4 in 0.9% NaCl, i.p., b.i.d.). Each mousewas treated from 8 to 13 weeks of age. Whole-body plethysmography wasperformed during week 12 and animals were subsequently killed andtissues harvested for TrkB Western blots and BDNF ELISA during week 13.

Statistical Analyses

Comparison of respiratory parameters between Wt and Het animals in theinitial phenotyping experiments were performed using unpaired Student'st test. Comparisons among Wt vehicle-treated, Wt drug-treated, Hetvehicle-treated, and Het drug-treated groups in the LM22A-4 trials,including plethysmography and Western blots were performed using one-wayANOVA with post hoc Least Significant Difference test (LSD) forintergroup comparisons. Results were considered significant if thep-value was <0.05. Data are presented as the mean±SEM.

Results

Development of Respiratory Dysfunction in Het Mice

The development of respiratory dysfunction in heterozygousMecp2^(tm1.1Jae) mice has not previously been described. Significantdifferences in breathing between Wt and Het mice first appeared between8 and 10 weeks after birth (FIG. 8). At 10 weeks, Het mice exhibited anabnormally high breathing frequency associated with marked decreases inexpiratory time (T_(e)) and total breath duration (T_(tot)) and a smallbut significant decrease in inspiratory time (Ti; FIG. 8A1-A4).Significant differences in respiratory frequency, T_(e) and T_(tot) (butnot T_(i)) persisted at 12 weeks. The number of apneas in the Hetpopulation increased between 8 and 12 weeks of age (FIG. 8B 1). This wasdue to a progressive increase in the proportion of Hets exhibitingsignificantly more apneas than Wt (20% at 8 weeks vs. 50% at 12 weeks).

BDNF Expression Deficits in the Brainstem of Het Mice

Mecp^(2tm1.1Jae)-null mice exhibit deficits in BDNF expression instructures critical for respiratory control, including the cranialsensory nodose ganglion (NG) and brainstem. To determine whether thedevelopment of respiratory dysfunction in Het mice is associated withchanges in BDNF expression, we compared BDNF protein levels in theseregions in Wt and Het mice at 8, 10, and 12 weeks of age (FIG. 9). BDNFlevels in the Het NG were significantly below Wt at all 3 ages (FIG.9A). Accordingly, we found reduced immunostaining for BDNF in themedullary nucleus tractus solitaries (nTS), the primary target ofafferent projections from NG sensory neurons to the brainstem (FIG. 9D;12 weeks of age). Despite this selective deficit within the Het nTS,differences in BDNF level between the Wt and Het medulla as a whole wereonly detectable by ELISA at 8 weeks of age (FIG. 9B). In the Het pons,the level of BDNF was normal at 8 weeks and fell below Wt values at 10and 12 weeks (FIG. 9C).

Improved Respiratory Function Following LM22A-4 Treatment

In light of our finding that Het mice exhibit BDNF deficits in the nTSand pons, regions in which BDNF/TrkB signaling is important forrespiratory control, we next sought to examine whether or not systemicadministration of LM22A-4, a small molecule BDNF loop domain mimeticthat acts as a selective TrkB agonist, could improve the Het breathingphenotype. To determine the brain penetrance of LM22A-4 followingsystemic administration, brainstem and forebrain samples were analyzedby LC MS/MS 1 h after a single intraperitoneal injection of 50 mg/kgLM22A-4 as described under Materials and Methods. These experimentsdemonstrated brain tissue concentrations (corrected for bloodcontamination) of 2.9 and 4.1 nM LM22A-4, respectively, which is wellwithin the range at which LM22A-4 exhibits biological activity in assaysof TrkB function. To evaluate the effects of systemic LM22A-4administration on respiratory function, we used whole-bodyplethysmography to compare resting ventilation in 12-week-old Wtvehicle-treated, Wt drug-treated, Het vehicle-treated and Hetdrug-treated animals following 4 weeks of twice daily injections ofLM22A-4 (50 mg/kg, i.p.) (FIG. 10A,B). In contrast to vehicle-treatedHet controls, LM22A-4-treated Hets exhibited values of breathingfrequency, T_(e) and T_(tot) that were not significantly different fromthose of Wt (FIG. 10A,B; pooled results of 4 independent experiments).The percentage of apneic animals was unaffected by drug treatment (datanot shown), and there were no significant differences in respiratoryfunction between vehicle- and drug-treated Wt mice. Moreover, drugtreatment had no effect on body weight in either Wt or Het micethroughout the treatment period (FIG. 10C).

LM22A-4 Treatment Reverses TrkB Phosphorylation Deficits in theBrainstem of Het Mice

We next sought to determine whether or not improved respiratory functionin LM22A-4-treated Hets is associated with increased TrkB signaling inthe brainstem. To address this issue, we used Western blots to comparethe ratio of phosphorylated TrkB to total full-length TrkB(p-TrkB^(Y817)/TrkB) in 13-week-old Wt vehicle-treated, Wt drug-treated,Het vehicle-treated, and Het drug-treated animals following 5 weeks ofsystemic treatment with LM22A-4 (50 mg/kg, i.p., b.i.d.) (FIG. 11). Tocontrol for possible changes in total TrkB levels between Wt and Hetsamples, or with LM22A-4 treatment, we also compared the ratios ofp-TrkB^(Y817), total full-length TrkB and total truncated TrkB to actin(p-TrkBY817/actin, TrkB/actin, TrkB-T/actin, respectively). Pons andmedulla samples from vehicle-treated (control) Hets exhibitedsignificant decreases in p-TrkBY817/TrkB and p-TrkBY817/actin comparedwith vehicle treated (control) Wt animals with no change in the levelsof total full-length TrkB or truncated TrkB. These deficits in TrkBphosphorylation in Het mice were completely reversed by treatment withLM22A-4 (FIG. 11). In fact, drug-treated Hets showed significantlyhigher levels of p-TrkB/TrkB and p-TrkB/actin than wild-type controls inthe pons. We also examined whether or not genotype and drug effects onTrkB phosphorylation were reflected in changes in phosphorylation of theserine/threonine protein kinase AKT (p-AKT) and extracellular signalregulated kinase ERK/MAPK (p-ERK), key downstream mediators of thebiological effects of TrkB activation. These experiments demonstratedsignificant decreases in p-AKT/AKT and p-AKT/actin in the pons of Hetmice compared with Wt controls that were reversed by LM22A-4 treatment,with no change in total AKT levels (FIG. 12). In contrast, there was nosignificant effect of genotype or drug treatment on the level ofp-ERK/ERK in pons samples from Wt and Het mice. In the medulla, althoughp-AKT/AKT and p-AKT/actin tended to be lower in Het mice compared withWt, these trends were not statistically significant. p-ERK/ERK in themedulla was significantly lower in Het animals compared with Wt controlsin both vehicle- and drug treated animals (FIG. 13).

The present finding that pharmacologic activation of TrkB in Mecp2 Hetmice eliminates respiratory tachypnea supports the role of BDNF/TrkBsignaling deficits in respiratory dysfunction in RTT and providesproof-of-concept for the therapeutic potential of TrkB agonists toimprove this and other aspects of the disease. In summary, our datademonstrate the ability of a BDNF loop domain mimetic to enhance TrkBactivation and restore wildtype respiratory frequency in Mecp2 Het mice.These findings provide validation of TrkB as a therapeutic target inmouse models of RTT and indicate that BDNF loop domain mimetics can beeffective at overcoming functional deficits associated with reduced BDNFexpression.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention, the following is claimed:
 1. A method oftreating Rett syndrome in a subject in need thereof, the methodcomprising administering to said subject a sub-anesthetic bolusinjection of ketamine at a dosage of 1-20 mg/kg.
 2. The method of claim1, further comprising administering a TrkB agonist in combination withthe NMDAR antagonist.
 3. The method of claim 2, wherein the TrkB agonistN,N′,N″Tris(2-hydroxyethyl)-1,3,5-benzenetricarboxamide or apharmaceutically effective salt thereof.
 4. The method of claim 2,wherein the NMDAR antagonist ketamine and the TrkB agonist isN,N′,N″Tris(2-hydroxyethyl)-1,3,5-benzenetricarboxamide or apharmaceutically effective salt thereof.